AU2001245407B2 - G12L, a gene associated with the thermal response - Google Patents

Description

1 G12L, A NOVEL GENE ASSOCIATED WITH THE THERMAL RESPONSE FIELD OF THE INVENTION The invention generally relates to nucleic acids and polypeptides encoded therefrom.

More specifically, the invention relates to nucleic acids encoding novel polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.

BACKGROUND OF THE INVENTION

OBESITY

Obesity is the most prevalent metabolic disorder in the United States affecting on the order of 35% of adults at an estimated cost of 300,000 lives and $70 billion in direct and indirect costs. As an epidemic it is growing due to the increase in the number of children who can be considered overweight or obese. Obesity is defined as an excess of body fat, frequently resulting in a significant impairment of health. Obesity results when adipocyte size or number in a person's body increases to levels that may result in one or more of a number of physical and psychological disorders. A normal-sized person has between 30 and 35 billion fat cells. When a person gains weight, these fat cells increase in size at first and later in number. Obesity is influenced by genetic, metabolic, biochemical, psychological, and behavioural factors. As such, it is a complex disorder that must be addressed on several fronts to achieve lasting positive clinical outcome.

Obese individuals are prone to ailments including: type II diabetes mellitus (NIDDM), hypertension, coronary heart disease, hypercholersterolemia, osteoarthritis, gallstones, cancers of the reproductive organs, and sleep apnea. Sleep apnea is episodes of not breathing during sleep that correlates with higher incidence of stroke and heart attack, two other factors contributing to obesity-linked morbidity and mortality among the clinically obese.

There are several well established treatment modes ranging from non-pharmaceutical to pharmaceutical intervention. Non-pharmaceutical intervention includes diet, exercise, psychiatric treatment, and surgical treatments to reduce food consumption or remove fat, Eilyw/keep/retype/P46923 pgs 1, 4 4a WO 01/64884 PCT/US01/06839 liposuction. Appetite suppressants and energy expenditure/nutrient-modifying agents represent the main focus of pharmacological intervention. Dexfenfluramine (REDUX®) and sibutramine (MERIDIA®) in the first class and beta3-adrenergic agonists and orlistat (XENICAL®) representing the latter.

Animal models have provided strong evidence that genetic make-up is influential in the determining the nature and extent of obesity. Though what is true in animals may not be true for humans, 40-80% of variation in body mass index (BMI, a measure of obesity correlating weight and height) can be attributed to genetic factors. While human obesity does not generally follow a Mendelian inheritance pattern, there are several rodent models that do so (Spiegelman, Cell.

1996 Nov 1;87(3):377-89; Weigle, Bioessays. 1996 Nov;18(11):867-74). As human obesity is a complex trait, it is therefore not surprising that single mutations in rodent might not be representative of causation in the majority of obese humans, though there are examples of humans with genetic lesions analogous to those found in rodents (Montague, Nature. 1997 Jun 26;387(6636):903-8; Clement, Nature. 1998 Mar 26;392(6674):398-401). Interestingly, animal models for complex phenotypes, such as hypertension and stroke, are also obese. This suggests that these animals may represent a more telling model for understanding the complexities of human obesity.

There are several rodent models of obesity that result in the inheritance of a single genetic lesion. Monogenetic obesity syndromes in mice that are well characterized but rarely, if ever, observed in humans include: obese aberrant termination of the translation of the satiety factor leptin. Mutations of the leptin receptor results in the obese diabetic mouse (db).

Agouti (Ay) is a coat color mutant that is obese. Normally only expressed in the skin, in the mutant animals it is ubiquitously expressed and may antagonize the binding ofmelanocyte stimulating hormone (MSH). MSH is derived from adrenocorticotropic hormone (ACTH) a major pituitary hormone that results from the proteolytic processing of the pro-hormone proopiomelanocortin (POMC). The fat phenotype is the consequence of a mutation in the hypothalamic pro-hormone converting enzyme carboxypeptidase E. The least well characterized obese mouse mutant is tub. tub encodes a cytosolic protein that may influence the processing of hypothalamic neuropeptide hormones such as neuropeptide Y (NPY, an appetite stimulating hormone) and POMC. Recently, a POMC knockout mouse was reported that has a phenotype analogous to several mouse models for obesity, particularly that of Ay. The POMC knockout has early onset obesity and has yellow hair color as well as adrenal insufficiency due to the apparent morphological absence of their adrenal gland. As there is no detectable corticosterone in these animals and corticosterones increase food intake, it is surprising that they WO 01/64884 PCT/US01/06839 are obese. The obese phenotype can be treated with alpha-MSH, a peptide hormone derived from POMC (Yaswen, Nat Med. 1999 Sep;5(9):1066-70).

Other animal models include faifa (fatty) rats, which bear many similarities to the ob/ob and db/db mice, discussed above. One difference is that, while fa/fa rats are very sensitive to cold, their capacity for non-shivering thermogenesis is normal. It is well established that thermogenesis and metabolism are closely coupled endocrinologically. Torpor, a condition analogous to hibernation and lethargy, seems to play a larger part in the maintenance of obesity in fa/fa rats than in the mice mutants. Further, several desert rodents, such as the spiny mouse, do not become obese in their natural habitats, but do become so when fed on standard laboratory feed.

BROWN ADIPOSE TISSUE Brown Adipose Tissue (BAT), also known as multilocular adipose tissue, is so called because of the its color due to the large number of capillaries and mitochondria in the cells making up this tissue. BAT is found primarily in the shoulder region and flanks of human embryo and newborn and then disappears in the first months of life. In animals, particularly hibernating animals and rodents, it is more abundant. BAT has features of an endocrine organ in that it is vascularized with capillaries and can receive direct sympathetic innervation.

Sympathetic neurotransmission leads to the release of the catecholamines noradrenaline and adrenaline resulting in the activation of a hormone-sensitive lipase. This results in the hydrolysis oftriglycerides that are converted to fatty acids and glycerol leading to an increase in oxygen consumption and heat production by uncoupling of the mitochondrial proton gradient from the formation of ATP via the activity of uncoupling proteins (UCPs). BAT stimulation by catecholamines results in non-shivering thermogenesis.

Evidence of BAT as an endocrine organ comes from the work of Himms-Hagen done in the late 1960's. Experiments involving the removal of BAT from rats acclimated to different temperatures and the effects upon enhanced calorigenic response to catecholamines lead to the following observations i) removal of interscapular BAT (IBAT) from cold-acclimated rats has no immediate effect on the calorigenic response of rats to catecholamines. The significance being that BAT is not the organ directly responsive to this stimulus, ii) With time (days) there is a progressive loss of the enhanced catecholamines response by rats that have had their IBAT removed, suggesting that BAT is responsible for the long-term maintenance of the catecholamines-induced thermogenic response. Interestingly, the ability of IBAT to maintain the enhanced response correlated with the duration of exposure to cold. This suggests that BAT has short term and long term effects on acclimation. With long-term cold acclimation there may 4 be a proliferation of BAT into regions other than that occupied by IBAT, thus maintaining the catecholamines response. Other work showing that transplantation of IBAT from animals acclimated to cold into those raised in the warm can confer a thermaogenic response under condition that normally would not also supports the endocrine nature of BAT.

The role of this endocrine organ in the maintenance of body weight as well as thermogenesis was demonstrated by the ablation of BAT in a transgenic mouse model using a BAT-specific promoter (UCP 1) controlling the expression of diphtheria toxin during the development of this tissue. Animals were found to be unable to maintain core body temperature when exposed to the cold, were hyperphagic, leptin resistant, and obese. The significance of this latter observation is that in the absence of BAT the mie have increased metabolic efficiency, due to the loss of BAT thermogenesis and/or BAT derived hormones. That is to say, in the absence of BAT and UCP, there is net accumulation of energy stored in the form of fat. Finally, in the case of one strain of mice with only a transient ablation of BAT, the metabolic defect is ameliorated with the reemergence of BAT [Lowell, Nature. 1993 Dec 23-30;366(6457):740-2; Friedman, Nature. 1993 Dec 23-30;366(6457)720-1]. These data taken together support the contention that BAT is a unique thermogenic and endocrine organ with a pivotal role in the metabolic status of organisms in which it is observed.

It is well established that endocrine organs regulate metabolism and in doing so must, perforce, regulate gene expression. By understanding the mechanism by which BAT provides endocrine and thermogenic activity, insights into the regulation of metabolism are gained. Genes and proteins associated with BAT thermogenesis, proliferation, or differentiation, will provide novel tools to treat or diagnose metabolic or other disorders. Furthermore, they may be used in screening efforts to uncover pharmaceuticals or molecules to treat metabolic and other disease, and can be valuable markers in uses such as pharmacogenomics.

In the claims of this application and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the words "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

Emilyw/keep/retype/P46923 pgs 1, 4 4a WO 01/64884 PCT/US01/06839 In one aspect, the invention provides an isolated gl2L nucleic acid molecule encoding a gl2L polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ ID NOS:1 or 3. In some embodiments, the gl2L nucleic acid molecule can hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a gl2L nucleic acid sequence. The invention also includes an isolated nucleic acid that encodes a gl2L polypeptide, or a fragment, homolog, analog or derivative thereof. For example, the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NOS:2 or 4. The nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS:1 or 3.

Also included in the invention is an oligonucleotide, an oligonucleotide which includes at least 6 contiguous nucleotides of a gl2L nucleic acid SEQ ID NOS:1 or 3) or a complement of said oligonucleotide.

Also included in the invention are substantially purified gl2L polypeptides (SEQ ID NO:2 or In some embodiments, the gl2L polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human gl2L polypeptide.

The invention also features antibodies that immunoselectively-bind to gl2L polypeptides.

In another aspect, the invention includes phannaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceuticallyacceptable carrier. The therapeutic can be, a gl2L nucleic acid, a gl2L polypeptide, or an antibody specific for a gl2L polypeptide. In a further aspect, the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.

In a further aspect, the invention includes a method of producing a polypeptide by culturing a cell that includes a gl2L nucleic acid, under conditions allowing for expression of the gl2L polypeptide encoded by the DNA. If desired, the gl2L polypeptide can then be recovered.

In another aspect, the invention includes a method of detecting the presence of a gl2L polypeptide in a sample. In the method, a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound. The complex is detected, if present, thereby identifying the gl2L polypeptide within the sample.

The invention also includes methods to identify specific cell or tissue types based on their expression of a gl2L.

WO 01/64884 PCT/US01/06839 Also included in the invention is a method of detecting the presence of a gl2L nucleic acid molecule in a sample by contacting the sample with a gl2L nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a gl2L nucleic acid molecule in the sample.

In a further aspect, the invention provides a method for modulating the activity of a gl 2L polypeptide by contacting a cell sample that includes the gl2L polypeptide with a compound that binds to the gl2L polypeptide in an amount sufficient to modulate the activity of said polypeptide. The compound can be, a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.

Also within the scope of the invention is the use of a Therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, metabolic disorders and diseases, such as obesity, diabetes, and cachexia; cell proliferative disorders and diseases, such as hyperplasia, cancer, and restenosis; neurodegenerative disorders and diseases, such as Alzheimer's Disease, multiple sclerosis and Parkinson's Disorder; immune disorders and diseases, such as AIDS, inflammation, and autoimmune diseases; and hematopoietic disorders and diseases, such as SCID, cyclic neutropenia, and thrombocythemia. In a preferred embodiment, the Therapeutic is used in the manufacture of a medicament for treating obesity and obesity-related disorders. Obesity-related disorders include, but are not limited to, type II diabetes mellitus (NIDDM), hypertension, coronary heart disease, hypercholesterolemia, osteoarthritis, gallstones, cancers of the reproductive organs, and sleep apnea. The Therapeutic can be, a gl2L nucleic acid, a gl2L polypeptide, or a gl2L-specific antibody, or biologically-active derivatives or fragments thereof.

The invention further includes a method for screening fot a modulator of disorders or syndromes including, metabolic disorders and diseases, such as obesity, diabetes, and cachexia; cell proliferative disorders and diseases, such as hyperplasia, cancer, and restenosis; neurodegenerative disorders and diseases, such as Alzheimer's Disease and Parkinson's Disorder; immune disorders and diseases, such as AIDS, inflammation, and autoimmune diseases; and hematopoietic disorders and diseases, such as SCID, cyclic neutropenia, and thrombocythemia. In a preferred embodiment, a method screens for a modulator of obesity and obesity-related disorders. The method includes contacting a test compound with a gl2L polypeptide and determining if the test compound binds to the gl2L polypeptide. Binding of the test compound to the gl2L polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.

WO 01/64884 PCT/US01/06839 Also within the scope of the invention is a method for screening for a modulator of activity, or of latency or predisposition to an disorders or syndromes including, metabolic disorders and diseases, such as obesity, diabetes, and cachexia; cell proliferative disorders and diseases, such as hyperplasia, cancer, and restenosis; neurodegenerative disorders and diseases, such as Alzheimer's Disease and Parkinson's Disorder; immune disorders and diseases, such as AIDS, inflammation, and autoimmune diseases; and hematopoietic disorders and diseases, such as SCID, cyclic neutropenia, and thronmbocythemia, by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes. In a preferred embodiment, the test animal has an increased risk of obesity and obesity-related disorders. The test animal expresses a recombinant polypeptide encoded by a gl2L nucleic acid. Expression or activity of gl2L polypeptide is then measured in the test animal, as is expression or activity of the protein in a control animal which recombinantly-expresses gl2L polypeptide and is not at increased risk for the disorder or syndrome. Next, the expression of gl2L polypeptide in both the test animal and the control animal is compared. A change in the activity of gl2L polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of the disorder or syndrome.

In yet another aspect, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a gl2L polypeptide, a gl2L nucleic acid, or both, in a subject a human subject). The method includes measuring the amount of the gl2L polypeptide in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the gl2L polypeptide present in a control sample. An alteration in the level of the gl2L polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject. Preferably, the predisposition includes, metabolic disorders and diseases, such as obesity, diabetes, and cachexia; cell proliferative disorders and diseases, such as hyperplasia, cancer, and restenosis; neurodegenerative disorders and diseases, such as Alzheimer's Disease and Parkinson's Disorder; immune disorders and diseases, such as AIDS, inflammation, and autoimmune diseases; and hematopoietic disorders and diseases, such as SCID, cyclic neutropenia, and thrombocythemia. In more preferred embodiments, the new polypeptides of the invention can be used in a method to screen for the presence of or predisposition to obesity and obesity-related disorders. Also, the expression levels of the new polypeptides of the invention can be used in a method to screen for the presence of or predisposition to various cancers, breast cancer.

In a further aspect, the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a gl2L polypeptide, a gl2L nucleic acid, or a gl2L-specific antibody to a subject a human WO 01/64884 PCT/US01/06839 subject), in an amount sufficient to alleviate or prevent the pathological condition. In preferred embodiments, the disorder, includes, metabolic disorders and diseases, such as obesity, diabetes, and cachexia; cell proliferative disorders and diseases, such as hyperplasia, cancer, and restenosis; neurodegenerative disorders and diseases, such as Alzheimer's Disease and Parkinson's Disorder; immune disorders and diseases, such as AIDS, inflammation, and autoimmune diseases; and hematopoietic disorders and diseases, such as SCID, cyclic neutropenia, and thrombocythemia. In more preferred embodiments, administration is to a subject suspected of suffering from obesity or obesity-related disorders.

In yet another aspect, the invention can be used in a method to identity the cellular components that interact with the gl2L nucleic acids and polypeptides, including cellular receptors and downstream effectors, by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, coprecipitation with antibodies or other specific-interacting molecules.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWING FIG. 1. Multiple sequence analysis shows that the Spot 14's and the gl2's are distinct, but related families of proteins. The boxed area is the location of the "Spot Box" and the underlined area indicates the "acid box" for the two gene families. mspotl4 mouse Spot 14 (GenBank X95279; SEQ ID NO:5), mspotl4 rat Spot 14 (GenBank NM_003251; SEQ ID NO:6), hsspotl4 human Spot 14 (GenBank Y08409; SEQ ID NO:7), hsdrgl21ike human gl2L (SEQ ID NO:2), mmdrgl21ike mouse gl2L (SEQ ID NO:4), and drgl2 zebrafish g12 (GenBank U27121; SEQ ID NO:8).

FIG. 2. TaqMan time course of mouse gl2L modulation.

FIG. 3. Tissue distribution of mouse gl2L at 48hrs of acclimation at 4C, 22C, and 33C by TaqMan analysis.

FIG. 4. Relative protein homology of Spot 14's, zebrafish g12, and human and mouse gl2L's.

FIG. 5. Relative cDNA homology of Spot 14's, zebrafish g12, and human and mouse gl2L's.

WO 01/64884 PCT/US01/06839 FIG. 6. Radiation Hybrid map of mouse gl2L. Mouse gl2L is located near the centromere of chromosome X. Radiation hybrid mapping vectors were submitted to Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the mouse genome with a lod score cut off of 14. The data format was the Whitehead Order using 93 hybrids.

(http://www.genome.wi.mit.edu/cgi-bin/mouse rh/rhmap-auto/rhmapper.cgi.) iOq0-90.5 is the band identifier for mouse gl2L. Cybb is cytochrome b 245 (GenBank U43384) and Xkh (GenBank AF155511) is the mouse homologue of the XK gene. Scale is 71.5 cR per cM.

DETAILED DESCRIPTION By identifying genes expressed in Brown Adipose Tissue (BAT) under conditions that affect the metabolic activity and proliferative status of BAT, the inventors have isolated novel nucleic acids and polypeptides with substantial homology to known nucleic acids and proteins.

One such homologous protein is Spot 14.

Spotl4 in human, mouse, and rat, are nuclearly localized proteins of approximately -18kD that are highly conserved between mouse, rat, and human. The human gene is 81% identical at the amino acid level to the rat Spotl4 gene and the mouse homolog is 94% identical to the rat gene product (Grillasca, FEBSLett. 1997 Jan 13;401(1):38-42). Rat Spotl4 has been relatively well studied and has been shown to have its expression modulated under a number of conditions relevant to metabolic regulation in WAT (White Adipose Tissue), BAT (insulin, retinoic acid, cAMP), and liver. It is up-regulated in liver by insulin, carbohydrates, and thyroid hormone This latter observed modulation lead to its identification as a TH-responsive gene in liver. Whereas insulin, TH, and carbohydrates up-regulate the expression of Spot 14, dietary fats and polyunsaturated fatty acids result in its down-regulation (Clarke, JNutr. 1990 Feb;120(2):225-31; Jump, Proc NatlAcad Sci USA. 1993 Sep 15;90(18):8454-8). Spot 14, given its expression pattern and modulation in response to metabolism influencing compounds, likely is playing an important role in lipogenesis and/or the development/proliferation of lipogenic tissues under normal and disease conditions (Grillasca, FEBSLett. 1997 Jan 13;401(1):38-42).

Spot 14 amplification is associated with cancer. Enhanced long-chain fatty acid synthesis may occur in breast cancer, where it is necessary for tumor growth and predicts a poor prognosis. The Spot 14 protein functions to activate genes encoding the enzymes of fatty acid synthesis. Amplification of chromosome region 1 lql3, where the human Spotl4 gene resides, also predicts a poor prognosis in breast tumors. Human Spot 14 gene is localized between markers D11S906 and D11S937, at the telomeric end of the amplified region at 1 lq13, and found that it was amplified and expressed in breast cancer-derived cell lines. Other findings WO 01/64884 PCT/US01/06839 supported a role for the protein as a determinant of tumor lipid metabolism. Expression of Spot 14 provided a pathophysiologic link between 2 prognostic indicators in breast cancer: enhanced lipogenesis and 11q13 amplification (Moncur, Cytogenet Cell Genet. 1997;78(2):131-2; Cunningham, Thyroid. 1998 Sep;8(9):815-25; Moncur, Proc NatlAcad Sci USA. 1998 Jun 9;95(12):6989-94).

Spot 14 proteins are believed to by the mammalian homologs of a small Zebrafish (Danio rerio) gastrulation-specific protein, g12 (Grillasca, FEBSLett. 1997 Jan 13;401(1):38- 42). Spot 14 has a high degree of homology to g12. The Spot protein and closely related proteins may, as a larger family of gl2-like proteins, play a role in differentiation during the development and/or proliferation oflipogenic (and other) tissues under normal and diseased conditions. The putative family members have several features in common: low molecular weight, a "spot box", and acidic pi's that are probably due to the presence of highly acidic regions of the carboxyterminus of 10-20 amino acids with high aspartic acid and glutamic acid content (Grillasca, FEBSLett. 1997 Jan 13;401(1):38-42).

The invention is based, in part, upon the discovery of a novel mouse gene, as well as its human homolog, encoding novel polypeptides that are more similar to g1 2 than the Spot 14 proteins from mouse, rat, and human. These novel genes and polypeptides are collectively designated herein as "gl2-like" (gl2L).

The novel gl2L nucleic acids of the invention include the nucleic acids whose sequences are provided in Tables 1A and 2A, or a fragment thereof. The invention also includes a mutant or variant gl2L nucleic acid, any of whose bases may be changed from the corresponding base shown in Tables 1A and 2A while still encoding a protein that maintains the activities and physiological functions of the gl2L protein fragment, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including complementary nucleic acid fragments. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way ofnonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.

These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to 20% or more of the bases may be so changed.

The novel gl2L proteins of the invention include the protein fragments whose sequences are provided in Tables 1B and 2B. The invention also includes a gl2L mutant or variant protein, any of whose residues may be changed from the corresponding residue shown in Tables 1B and WO 01/64884 PCT/US01/06839 2B while still encoding a protein that maintains its native activities and physiological functions, or a functional fragment thereof. I1 the mutant or variant gl2L protein, up to 20% or more of the residues may be so changed. The invention further encompasses antibodies and antibody fragments, such as Fab or (Fab)2, that bind immunospecifically to any of the gl2L proteins of the invention.

Human gl2L Table 1A describes the nucleic acid sequence of a nucleotide fragment encoding a human gl2L protein. Putative ATG start codon is labeled in bold underline type. Putative stop codon is in bold italic underline type. Sequence is presented 45 characters per line.

WO 01/64884 PCTIUSO1/06839 Table IA. Human gl2L nucleotide fragment (SEQ IUD NO: 1).

1 GCGCGCCAGGGGTGGCCCTGAGCGCCGGCGACACCTTTCCTGGAC 46 TATATAATTGAGCACCTGGGATGGGTAGGGGGCCAACGCAGTCACC 91 GCCGTCCGCAGTCACAGTCCAGCCACTGACCGCAGCAGCGCCCTT 136 GCGTAGCAGCCGCTTGCAGCGAGAACACTGAATTGCCAACGAGCA 181 GGAGAGTCTCAAkGGCGCAAGAGGAGGCCAGGGCTCGACCCACAGA 226 GCACCCTCAGCCATCGCGAGTTTCCGGGCGCCAAGCCAGGAGAA 271 GCCGCCCATCCCGCAGGGCCGGTCTGCCAGCGAGACGAGAGTTGG 316 CGAGGCGGAGGAGTGCCGGGAATCCCGCCCACACCGGCTATAGCCC 361 AGGCCCCCAGCGCGGGCCTTGGAGAGCGCGTGAAGGCGGGCATCC 406 CCTTGACCCGGCCGACCATCCCCGTGCCCCTGCGTCCCTGCGCTC 451 CAACGTCCGCGCGGCCACCATGATGCAAATCTGCGACACCTACAA 496 CCAGAAGCACTCGCTCTTTAACGCCATGAATCGCTTCATTGGCGC 541 CGTGAACAACATGGACCAGACGGTGATGGTGCCCAGCTTGCTGCG 586 CGACGTGCCCC'TGGCTGACCCCGGGTTAGACAACGAGGTCACCGT 631 GGAGGTAGGCCGCAGTGGCAGCTGCCTGGAGGAGCGCACGACCCC 676 GGCCCCAAGCCCGGGCAGCGCCAATGGAAGCTTTTTCGCGCCCTC 721 CCGGGACATGTACAGCCACTACGTGCTGCTCAAGTCCATCCGCAA 766 CGATATTGAGTGGGGAGTCCTGCACCAGCCGCCTCCACCGGCTGG 811 GAGCGAGCAGGGCAGTGCCTGGAAGTCCAAGGACATCCTGGTGGA 856 CCTGGGCCACTTGGAGGGTGCGGACGCCGGCGAAGAAGACCTGGA 901 ACAGCAGTTCCACTACCACCTGCGCGGGCTGCACACTGTGCTCTC 946 GAAACTCACGCGCAAAGCCAACATCCTCACTAACAGATACAAGCA 991 GGAGATCGGCTTOGGCAATTGGGGCCACTGAGGCGTGGCGCCCGT 1036 GGCTGCCCAGCACCTTCTTCGACCCATCTCACCCTCTCTCATTCC 1081 TCAALAGCTTTTTTTTTTTCCCTGGCTGGGGGGCGGAAAGGGCAAA 1126 CTG WO 01/64884 PCTIUSO1/06839 A polypeptide encoded by SEQ ED NO:lI is presented using the one-letter code in Table lB.

Table 1B. Human gl2L polypeptide, sequence (SEQ ID NO:2).

1 MMQICDTYNQKHSLiFNAMNRF IGAVNNMDQTVMVPSLLRDVPLADPGLDNEVSVEVGGSGS CLEERTTPA 71. PSPGSNGSFFAPSRDMYSYVLLKSIRDIEWGVLlQPPPPAGSEEGSAWKSKDILVDLGULEGADAGE 141 EDLERQQFHYHLRGIHTVLSKLTRKZANILTNRYKQEIGFGNWGH Mouse g12L Table 2A describes the nucleic acid sequence of a nucleotide fragment encoding a mouse gl2L protein. Putative ATG start codon is labeled in bold underline type. Putative stop codon is in bold italic underline type. Putative polyadenylation site is in italics underline type.

Sequence is presented 45 characters per line.

Table 2A. Mouse gl2L nucleotide fragment (SEQ ID NO:3).

1 CGGACGCGTGGGGGAGGTAGGAGGAGGAGACATCAGGGGTGGTCC 46 TGGGCGCCTGGGACACCTTTCCCGGACTATAAATTGAGCACCTGG 91 AATGGGCAGGGGGCCGGAGCAACCACAGTCGCCCTTACTCACAGT 136 CCGATCAGTGACCGCAGCAGCGOCCTTGGGCAGCCACCGTCCGCA 181 ACGCA7AGCACTAGAACCAGGGGATTTCGCAGTGCAAGAGAAA 226 GGCTAGACCCAGCCACCCACCGTCAATCCTGAGCCA-AGATAAGA 271 GCAGCCGGGCCTCACGAAGGGCTGAGCTGAGAJXAGAAGCAAGTTA 316 GAGAGGGCGGAGAGGATCTGGGAATCCCGTCACACCGGCTTCAJA 361 GCAGGCTCCCGGCATCAGCCTCTGAGAGCGCTTGAXGGCGGCATC 406 GCCAGCGGTCTATCTCCGTGTACCAGCGTCCCTGTGTTTCCGCGC 451 CCGCTCGGCCACCATGATGCAAATCTGCGACACATATAACCAGAA 496 GCACTCGCTCTTTAACGCCATGJAT7CGCTTCATTGGCGCGGTGAA 541 CA7ACATGGACCAGACOGGATGGTGCCCAGTCTGCTGCGCGACGT 586 ACCCCTGTCCGAGCCGGAGATAGACGAGGTCAGCGTGGAGGTAGG WO 01164884 PCT/USOt/06839 631 CGGCAGTGGCGGCTGCCTGGAGGAGCGCACGACCCCGGCCCCAAG 676 CCCGGGCAGCGCCAATGAAAGCTTTTTCGCGCCCTCCCGGGACAT 721 GTACAGCCACTACGTGCTGCTCAAGTCCATCCGCAATGATATCGA 766 GTGGGGAGTCCTGCACCAGCCTTCGTCTCCGCCGGCCGGGAGCGA 811 GGAGAGCACCTGGAAGCCCAJAGGACATCCTGGTGGGCCTGAGTCA 856 CTTGGAGAGCGCGGATGCGGGCGAGGAAGATCTGGAGCAGCAGTT 901 CCACTACCACCTGCGCGGGCTGCACACCGTGCTCTCCAAACTCAC 946 CCGAAAAGCCAACATCCTCACCAATAGATACAAGCAGGAGATCGG 991 CTTCAGTAATTGG3GGCCACTcGAGGCGGGGGCTGTCCCCGCTGCCC 1036 AGCACCCTCTCTCGGGTCGGCTCTACCACCCCTCTCTTTCCTCCA 1081 AGCTATTTTCTTCCTGGTTGTGGGGCGCGAAGGGCACACTGTAAA 1126 GTTGGGCTGTGTACTTGGTGGGGTTAGTGTGGAGAAGAGGGCCTC 1171 ATCGCGAGAGCAGAGGAAAGTAGTCGCCAGAGAGGGGGGTTCAAA 1216 GACCCCCGGAGGGGGCCTACTCTGTGTTGGTGGGAATGGAACTGG 1261 GCCGATGTCCTTCATTCAGCCTGTGCCTTTCTTGGGGTTTCTTTT 1306 CTGTTTTTCTTTCCGGAAGAGAAGGGCCTGAGAAAGGGCCATGCC 1351 AGGGCACAGTGCTGGGTTGCCACACATGGGAGGGCAGCTTCTAGC 1396 CGGGTGCTTGGGGGAGGCGGGGCTCAGCCTCCTGCTGCCCTGCCT 1441 TGAGCTGCCAGAGGAGGCCTTGGCGTTGCTAGGATTGCGTCAGTT 1486 TTCCTGTTTGCACTATTTCTTTTTGTAACAGTGACCCTGTCTTAJA 1531 GTCTTTCAGATCTCTTTCCTTTGAAACTTCGTCGATTCCATTGTG 1576 ATAAGCGCACAAACAGCACTGTTGGTAACCGGTACTACTTTATTA 1621 ATGATTTTCTGTTACACTGTACAGTAGTCCTGTGGCACCCTATCC WO 01/64884 PCT/US01/06839 1666 CTTTCACGCCACCCCTCCCCCGCCCGTGTGTGTAAACTGGCGATG 1711 TGCCAGCTAGGATGAAGCTTGCCACTCGGCTAGCGAAAATAATTA 1756 ACATTATTATGAGAAAGTGGATTTATCTAAAGTGGAACCAGCTGA 1801 CATTATATCTGTATCGTATGGAGAATGATGAAGGGCTCCACTGTT 1846 GTTATATGTCTTGTTTATTTAAAACTTTTTTTAATCCAGATGTAG 1891 ACTATATTCTAAAAAATAAAAACGCAGATGTGTTAAC A polypeptide encoded by SEQ ID NO:3 is presented using the one-letter code in Table 2B.

Table 2B. Mouse gl2L polypeptide sequence (SEQ ID NO:4).

1 MMQICDTYNQKHSLFNAMNRFIGAVNNMDQTVMVPSLLRDVPLSEPEIDEVSVEVGGSGGCLEERTTPAP 71 SPGSANESFFAPSRDMYSHYVLLKSIRNDIEWGVLHQPSSPPAGSEESTWKPKDILVGLSHLESADAGEE 141 DLEQQFHYHLRGLHTVLSKLTRKANILTNRYKQEIGFSNWGH An alignment of the gl2L polypeptides with zebrafish g12 and the mouse, rat, and human Spot 14 polypeptides is shown in FIG. 1.

The gl2L nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in metabolic disorders and diseases, such as obesity, diabetes, and cachexia; cell proliferative disorders and diseases, such as hyperplasia, cancer, and restenosis; neurodegenerative disorders and diseases, such as Alzheimer's Disease and Parkinson's Disorder; immune disorders and diseases, such as AIDS, inflammation, and autoimmune diseases; and hematopoietic disorders and diseases, such as SCID, cyclic neutropenia, and thrombocythemia. In preferred embodiments, the nucleic acids and proteins of the invention are used in therapeutic applications for obesity-related disorders, other metabolic diseases, proliferative disorders, and other diseases. Obesity related disorders include, but are not limited to, type II diabetes mellitus (NIDDM), hypertension, coronary heart disease, hypercholesterolemia, osteoarthritis, gallstones, cancers of the reproductive organs, and sleep apnea. For example, a cDNA encoding human gl2L maybe useful in gene therapy, and the human gl2L protein may be useful when administered to a subject in need thereof. The novel nucleic acid encoding human gl2L protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the WO 01/64884 PCT/US01/06839 protein are to be assessed, diseases related to single nucleotide polymorphisms, or other mutations. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.

gl2L Nucleic Acids and Polypeptides One aspect of the invention pertains to isolated nucleic acid molecules that encode gl2L polypeptides or biologically-active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify gl2L-encoding nucleic acids gl2L mRNAs) and fragments foruse as PCR primers for the amplification and/or mutation of gl2L nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules cDNA or genomic DNA), RNA molecules mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double-stranded DNA.

A gl2L nucleic acid can encode a mature gl2L polypeptide. As used herein, a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an open reading frame described herein. The product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an open reading frame, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+1 to residue N remaining. Further as used herein, a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or WO 01/64884 PCT/US01/06839 phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.

The term "probes", as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorterlength oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.

The term "isolated" nucleic acid molecule, as utilized herein, is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.

Preferably, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid sequences located at the and 3'-tennini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated gl2L nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, kb or 0.1 kb ofnucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived brain, heart, liver, spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the invention, a nucleic acid molecule having the nucleotide sequence of SEQ ID NOS:1 or 3, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NOS:1 or 3 as a hybridization probe, gl2L molecules can be isolated using standard hybridization and cloning techniques as described in Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL 2 n d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley Sons, New York, NY, 1993.) A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to gl2L nucleotide sequences can be prepared by standard synthetic techniques, using an automated DNA synthesizer.

WO 01/64884 PCT/US01/06839 As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID NOS:1 or 3, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.

In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NOS:1 or 3, or a portion of this nucleotide sequence a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an gl2L polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NOS:1 or 3, is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOS:1 or 3, that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in SEQ ID NOS:1 or 3, thereby forming a stable duplex.

As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.

Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence.

Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the WO 01/64884 PCT/US01/06839 native compound but differs from it in respect to certain components or side chains. Analogs maybe synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.

Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below. Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley Sons, New York, NY, 1993, and below.

A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms ofgl2L polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for a gl2L polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human gl2L protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS:2 or 4, as well as a polypeptide possessing gl2L biological activity. Various biological activities of the gl2L proteins are described below.

A gl2L polypeptide is encoded by the open reading frame of a gl2L nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon.

An ORF that represents the coding sequence for a full protein begins with an ATG "start" codon and terminates with one of the three "stop" codons, namely, TAA, TAG, or TGA. For the WO 01/64884 PCT/US01/06839 purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a bonafide cellular protein, a minimum size requirement is often set, a stretch ofDNA that would encode a protein of 50 amino acids or more.

The nucleotide sequences determined from the cloning of the human gl2L genes allows for the generation of probes and primers designed for use in identifying and/or cloning gl2L homologues in other cell types, e.g. from other tissues, as well as gl2L homologues from other vertebrates. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ ID NOS:I or 3; or an anti-sense strand nucleotide sequence of SEQ ID NOS:1 or 3; or of a naturally occurring mutant of SEQ ID NOS:1 or 3.

Probes based on the human gl2L nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express a gl2L protein, such as by measuring a level of a gl2L-encoding nucleic acid in a sample of cells from a subject e.g., detecting gl2L mRNA levels or determining whether a genomic gl2L gene has been mutated or deleted.

"A polypeptide having a biologically-active portion of a gl2L polypeptide" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologicallyactive portion of gl2L can be prepared by isolating a portion of SEQ ID NOS:1 or 3, that encodes a polypeptide having a gl2L biological activity (the biological activities of the gl2L proteins are described below), expressing the encoded portion of gl2L protein by recombinant expression in vitro) and assessing the activity of the encoded portion of gl2L.

gl2L Nucleic Acid and Polypeptide Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NOS:1 or 3, due to degeneracy of the genetic code and thus encode the same gl2L proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS:I or 3. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS:2 or 4.

WO 01/64884 PCT/US01/06839 In addition to the human gl2L nucleotide sequences shown in SEQ ID NO:1, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the g12L polypeptides may exist within a population the human population). Such genetic polymorphism in the gl2L genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding a gl2L protein, preferably a vertebrate gl2L protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the gl2L genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the gl2L polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the gl2L polypeptides, are intended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding g 12L proteins from other species, and thus that have a nucleotide sequence that differs from the human sequence of SEQ ID NOS: 1, are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the gl2L cDNAs of the invention can be isolated based on their homology to the human gl2L nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.

Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1 or 3. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.

Homologs nucleic acids encoding gl2L proteins derived from species other than human) or other related sequences paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular humnan sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.

As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter WO 01/64884 PCT/US01/06839 sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium.

Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 0 C for short probes, primers or oligonucleotides 10 nt to 50 nt) and at least about 60 0 C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HC1 (pH 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65 0 C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50 0 C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequences of SEQ ID NOS:1 or 3, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature encodes a natural protein).

In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1 or 3, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55 0 C, followed by one or more washes in 1X SSC, 0.1% SDS at 37 0 C. Other conditions of moderate stringency that maybe used are well-known within the art. See, Ausubel ,et al. 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley Sons, NY, and Kriegler, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.

In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ID NOS:1 or 3 or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low WO 01/64884 PCT/US01/06839 stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HC1 (pH 5 inM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40'C, followed by one or more washes in 2X SSC, 25 mM Tris-HC1 (pH 5 mM EDTA, and 0.1% SDS at 50 0 C. Other conditions of low stringency that may be used are well known in the art as employed for cross-species hybridizations). See, Ausubel, et al. 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA 78: 6789-6792.

Conservative Mutations In addition to naturally-occurring allelic variants of gl2L sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO NOS:1 or 3, thereby leading to changes in the amino acid sequences of the encoded gl 2L proteins, without altering the functional ability of said gl2L proteins. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NOS:2 or 4. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the gl2L proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the gl2L proteins of the invention are predicted to be particularly nonamenable to alteration. Amino acids for which conservative substitutions can be made are wellknown within the art.

Another aspect of the invention pertains to nucleic acid molecules encoding gl2L proteins that contain changes in amino acid residues that are not essential for activity. Such gl2L proteins differ in amino acid sequence from SEQ ID NOS:2 or 4, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences of SEQ ID NOS:2 or 4. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ T) NOS:2 or 4; more preferably at least about 70% homologous to SEQ ID NOS:2 or 4; still more preferably at least about 80% homologous to SEQ ID NOS:2 or 4; even more preferably at least about 90% homologous to SEQ ID NOS:2 or 4; and most preferably at least about homologous to SEQ ID NOS:2 or 4.

An isolated nucleic acid molecule encoding a gl2L protein homologous to the protein of SEQ ID NOS:2 or 4, can be created by introducing one or more nucleotide substitutions, WO 01/64884 PCT/US01/06839 additions or deletions into the nucleotide sequence of SEQ ID NOS: 1 or 3, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.

Mutations can be introduced into SEQ ID NOS:2 or 4, by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains lysine, arginine, histidine), acidic side chains aspartic acid, glutamic acid), uncharged polar side chains glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains threonine, valine, isoleucine) and aromatic side chains tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in the gl2L protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a gl2L coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for gl2L biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NOS:2 or 4, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.

The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved "strong" residues or fully conserved "weak" residues. The "strong" group of conserved amino acid residues may be any one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other. Likewise, the "weak" group of conserved residues may be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM, IIFY, wherein the letters within each group represent the single letter amino acid code.

In one embodiment, a mutant gl2L protein can be assayed for the ability to the ability to affect adipose tissue differentiation or development. In yet another embodiment, a mutant gl2L protein can be assayed for the ability to regulate a more general biological function the metabolism of an animal.).

WO 01/64884 PCT/US01/06839 Antisense Nucleic Acids Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS: 1 or 3, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 100, 250 or 500 nucleotides or an entire gl2L coding strand, or to only a portion thereof.

Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of an gl2L protein of SEQ ID NOS:2 or 4; or antisense nucleic acids complementary to an gl2L nucleic acid sequence of SEQ ID NOS:1 or 3, are additionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a g12L protein. The tenn "coding region" refers to the region of the nucleotide sequence comprising codons that are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding the gl2L protein.

The term "noncoding region" refers to 5' and 3' sequences that flank the coding region that are not translated into amino acids also referred to as 5' and 3' untranslated regions).

Given the coding strand sequences encoding the gl2L protein disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of gl2L mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of gl2L nRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of gl2L mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids phosphorothioate derivatives and acridine substituted nucleotides can be used).

Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- WO 01/64884 PCT/US01/06839 acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, dihydrouracil, beta-D-galactosylqueosine, inosine, N6isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, oxyacetic acid wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, acid 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a gl2L protein to thereby inhibit expression of the protein by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol 111 promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of the invention is an aanomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific doublestranded hybrids with complementary RNA in which, contrary to the usual p-units, the strands run parallel to each other. See, Gaultier, et al., 1987. Nucl. Acids Res. 15: 6625-6641. The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (see, Inoue, WO 01/64884 PCT/US01/06839 et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (see, e.g., Inoue, etal., 1987. FEBSLett. 215: 327-330.

Ribozymes and PNA Moieties Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.

In one embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a singlestranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave gl2L mRNA transcripts to thereby inhibit translation of gl2L mRNA. A ribozyme having specificity for an gl2L-encoding nucleic acid can be designed based upon the nucleotide sequence of an gl2L cDNA disclosed herein SEQ ID NOS:1 or For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a gl2L-encoding mRNA. See, U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al. gl2L mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.

Alternatively, gl2L gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gl2L nucleic acid the gl2L promoter and/or enhancers) to form triple helical structures that prevent transcription of the gl2L gene in target cells. See, Helene, 1991. Anticancer Drug Des. 6: 569-84; Helene, et al. 1992. Ann. N.Y.

Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.

In various embodiments, the gl2L nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, Hyrup, et al., 1996. BioorgMed Chem 4: 5-23. As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics DNA mimics) in that the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide WO 01/64884 PCT/US01/06839 synthesis protocols as described in Hyrup, et al., 1996. supra; Perry-O'Keefe, et al., 1996. Proc.

Natl. Acad. Sci. USA 93: 14670-14675.

PNAs of gl2L can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, inducing transcription or translation arrest or inhibiting replication. PNAs of gl2L can also be used, for example, in the analysis of single base pair mutations in a gene PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, S1 nucleases (see, Hyrup, et al., 1996.supra); or as probes or primers for DNA sequence and hybridization (see, Hyrup, et al., 1996, supra; Perry-O'Keefe, et al., 1996.

supra).

In another embodiment, PNAs ofgl2L can be modified, to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA- DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.

For example, PNA-DNA chimeras of gl2L can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, etal., 1996. supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al., 1996. supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, phosphoramidite, can be used between the PNA and the 5' end of DNA. See, Mag, etal., 1989. Nucl AcidRes 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment. See, Finn, et al., 1996. supra. Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, Petersen, et al., 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.

In other embodiments, the oligonucleotide may include other appended groups such as peptides for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, Letsinger, et al., 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, Krol, et al., 1988. BioTechniques 6:958-976) or intercalating agents (see, Zon, 1988. Pharm. Res. WO 01/64884 PCT/US01/06839 539-549). To this end, the oligonucleotide may be conjugated to another molecule, a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridizationtriggered cleavage agent, and the like.

gl2L Polypeptides A polypeptide according to the invention includes a polypeptide including the amino acid sequence of gl2L polypeptides whose sequences are provided in SEQ ID NOS:2 or 4. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID NOS:2 or 4, while still encoding a protein that maintains its gl2L activities and physiological functions, or a functional fragment thereof.

In general, an gl2L variant that preserves gl2L-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.

One aspect of the invention pertains to isolated gl2L proteins, and biologically-active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-gl2L antibodies. In one embodiment, native gl2L proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, gl2L proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a gl2L protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

An "isolated" or "purified" polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the gl2L protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of gl2L proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the language "substantially free of cellular material" includes preparations of gl2L proteins having less than about 30% (by dry weight) of non-gl2L proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-gl2L proteins, still more preferably less than about 10% of non-gl2L proteins, and most preferably less than WO 01/64884 PCT/US01/06839 about 5% ofnon-gl2L proteins. When the gl2L protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the gl2L protein preparation.

The language "substantially free of chemical precursors or other chemicals" includes preparations of gl2L proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of gl 2L proteins having less than about 30% (by dry weight) of chemical precursors or non-gl2L chemicals, more preferably less than about 20% chemical precursors or non-gl2L chemicals, still more preferably less than about 10% chemical precursors or non-gl2L chemicals, and most preferably less than about 5% chemical precursors or non-gl2L chemicals.

Biologically-active portions of gl2L proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the gl 2L proteins the amino acid sequence shown in SEQ ID NOS:2 or 4) that include fewer amino acids than the full-length gl2L proteins, and exhibit at least one activity of a gl2L protein.

Typically, biologically-active portions comprise a domain or motif with at least one activity of the gl2L protein. A biologically-active portion of a gl2L protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.

Moreover, other biologically-active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native gl2L protein.

In an embodiment, the gl2L protein has an amino acid sequence shown in SEQ ID NOS:2 or 4. In other embodiments, the gl2L protein is substantially homologous to SEQ ID NOS:2 or 4, and retains the functional activity of the protein of SEQ ID NOS:2 or 4, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the gl2L protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NOS:2 or 4, and retains the functional activity of the gl2L proteins of SEQ ID NOS:2 or 4.

Determining Homology Between Two or More Sequences To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first WO 01/64884 PCT/US01/06839 sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").

The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMolBiol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NOS:1 or 3.

The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.

Chimeric and Fusion Proteins The invention also provides gl2L chimeric or fusion proteins. As used herein, a gl2L "chimeric protein" or "fusion protein" comprises a gl2L polypeptide opcrativcly-linkcd to a non-gl2L polypeptide. An "gl2L polypeptide" refers to apolypeptide having an amino acid sequence corresponding to an gl2L protein (SEQ ID NOS:2 or whereas a "non-gl2L polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the gl2L protein, a protein that is different from the gl2L protein and that is derived from the same or a different organism. Within a gl2L fusion protein the gl2L polypeptide can correspond to all or a portion of a gl2L protein. In one embodiment, a gl2L fusion protein comprises at least one biologically-active portion of an g12L protein. In another embodiment, a gl2L fusion protein comprises at least two biologically- WO 01/64884 PCT/US01/06839 active portions of a gl2L protein. In yet another embodiment, a gl2L fusion protein comprises at least three biologically-active portions of a gl2L protein. Within the fusion protein, the term "operatively-linked" is intended to indicate that the gl2L polypeptide and the non-gl2L polypeptide are fused in-frame with one another. The non-gl2L polypeptide can be fused to the N-terminus or C-terminus of the gl2L polypeptide.

In one embodiment, the fusion protein is a GST-gl2L fusion protein in which the gl2L sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant gl2L polypeptides.

In another embodiment, the fusion protein is a gl2L protein containing a heterologous signal sequence at its N-terminus. In certain host cells mammalian host cells), expression and/or secretion of gl2L can be increased through use of a heterologous signal sequence.

In yet another embodiment, the fusion protein is a gl2L-immunoglobulin fusion protein in which the gl2L sequences are fused to sequences derived from a member of the immunoglobulin protein family. The gl2L-irnmunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a gl2L ligand and a gl2L protein, to thereby suppress gl2L-mediated signal transduction in vivo. The gl2L-immunoglobulin fusion proteins can be used to affect the bioavailability of a gl2L cognate ligand. Inhibition of the gl2L ligand/ gl2L interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating promoting or inhibiting) cell survival. Moreover, the gl2L-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-gl2L antibodies in a subject, to purify gl2L ligands, and in screening assays to identify molecules that inhibit the interaction of gl2L with a gl2L ligand.

A gl2L chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley Sons, 1992). Moreover, many expression vectors are commercially WO 01/64884 PCT/US01/06839 available that already encode a fusion moiety a GST polypeptide). A gl2L-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the gl2L protein.

gl2L Agonists and Antagonists It is useful to test for compounds having the property of increasing or decreasing gl2L activity. This increase in activity may come about in a variety of ways, for example: by increasing or decreasing the copies of the gene in the cell (amplifiers and deamplifiers); by increasing or decreasing transcription of the gl2L gene (transcription up-regulators and downregulators); by increasing or decreasing the translation of gl2L mRNA into protein (translation up-regulators and down-regulators); or by increasing or decreasing the activity of gl2L itself (agonists and antagonists).

Compounds that are amplifiers and deamplifiers may be identified by contacting cells or organisms with the compound, and then measuring the amount of DNA present that encodes gl2L (Ausubel, Brent et al. 1987). Compounds that are transcription up-regulators and downregulators may be identified by contacting cells or organisms with the compound, and then measuring the amount of mRNA produced that encodes gl2L (Ausubel, Brent et al. 1987).

Compounds that are translation up-regulators and down-regulators may be identified by contacting cells or organisms with the compound, and then measuring the amount of gl2L polypeptide produced (Ausubel, Brent et al. 1987). Compounds that are agonists or antagonists may be identified by contacting cells or organisms with the compound, and then measuring gl2L activity; this may also be carried out in vitro.

The invention also pertains to variants of the gl2L proteins that function as either gl2L agonists mimetics) or as gl2L antagonists. Variants of the gl2L protein can be generated by mutagenesis discrete point mutation or truncation of the gl2L protein). An agonist of the gl2L protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the gl2L protein. An antagonist of the gl2L protein can inhibit one or more of the activities of the naturally occurring form of the gl2L protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade that includes the gl2L protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the gl2L proteins.

Variants of the gl2L proteins that function as either gl2L agonists mimetics) or as gl2L antagonists can be identified by screening combinatorial libraries of mutants truncation mutants) of the gl2L proteins for gl2L protein agonist or antagonist activity. In one WO 01/64884 PCT/US01/06839 embodiment, a variegated library of gl2L variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of gl2L variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential gl2L sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins for phage display) containing the set of gl2L sequences therein. There are a variety of methods that can be used to produce libraries of potential gl2L variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential gl2L sequences. Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, Narang, 1983. Tetrahedron 39: 3; Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al., 1984. Science 198: 1056; Ike, et al., 1983. Nucl. Acids Res. 11: 477.

Polypeptide Libraries In addition, libraries of fragments of the gl2L protein coding sequences can be used to generate a variegated population of gl2L fragments for screening and subsequent selection of variants of a gl2L protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a gl2L coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes of the gl2L proteins.

Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of gl2L proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can WO 01/64884 PCT/US01/06839 be used in combination with the screening assays to identify gl2L variants. See, Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al., 1993. Protein Engineering 6:327-331.

Anti-gl2L Antibodies The invention encompasses antibodies and antibody fragments, such as Fab or (Fab)2, that bind immunospecifically to any of the gl2L polypeptides of said invention.

The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab' and F(ab')2 fragments, and an Fab expression library. In general, antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGi, IgG 2 and others. Furthermore, in humans, the light chain maybe a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.

An isolated protein of the invention intended to serve as an antigen, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.

The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence shown in SEQ ID NOs:2 or 4, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.

In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of gl2L that is located on the surface of the protein, a hydrophilic region. A hydrophobicity analysis of the gl2L protein sequence will indicate which regions of a gl2L polypeptide are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody WO 01/64884 PCT/US01/06839 production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J Mol. Biol.

157: 105-142, each incorporated herein by reference in their entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.

A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.

Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.

1. Polyclonal Antibodies For the production ofpolyclonal antibodies, various suitable host animals rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels aluminum hydroxide), surface active substances lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. Additional examples of adjuvants that can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG WO 01/64884 PCT/US01/06839 fraction of immune serum. Subsequently, or alternatively, the specific antigen that is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of imnunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

2. Monoclonal Antibodies The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs thus contain an antigenbinding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].

Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRTdeficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which WO 01/64884 PCT/US01/06839 can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). It is an objective, especially important in therapeutic applications of monoclonal antibodies, to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.

After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, 1986). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mamnal.

The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalentlyjoining to the immunoglobulin coding sequence all or part WO 01/64884 PCT/US01/06839 of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

3. Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigenbinding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.

Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature. 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a nonhuman immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

4. Human Antibodies Fully human antibodies essentially relate to antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein.

Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using WO 01/64884 PCT/US01/06839 human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, mice in which the endogenous inununoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al.

(Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al,( Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).

Human antibodies may additionally be produced using transgenic non-human animals that are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication W094/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse T M as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells that secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.

An example of a method of producing a non-human host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent WO 01/64884 PCT/US01/06839 No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.

A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an imnunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.

Fah Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see U.S. Patent No. 4,946,778).

In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: an F(ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F, fragmeits.

6. Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two WO 01/64884 PCT/US01/06839 immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.

DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzvmology, 121:210 (1986).

According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g.

tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies or antibody fragments F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific WO 01/64884 PCT/US01/06839 antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab') 2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic ann of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule CD2, CD3, CD28, or B7), or Fe receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells that express a particular antigen. These antibodies possess an antigen-binding arm and an arm that binds a cytotoxic agent or a WO 01/64884 PCT/US01/06839 radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).

7. Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.

Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089).

It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.

Examples of suitable reagents for this purpose include iminothiolate and methyl-4mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.

8. Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191- 1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

9. Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope a radioconjugate).

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, WO 01/64884 PCT/US01/06839 mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production ofradioconjugated antibodies. Examples include 212Bi, 13, 131n 90, and 1 86Re.

Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).

Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody. See W094/11026.

In another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" avidin) that is in turn conjugated to a cytotoxic agent.

10. Immunoliposomes The antibodies disclosed herein can also be formulated as immunoliposomes.

Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.

Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst., 81(19): 1484 (1989).

WO 01/64884 PCT/US01/06839 11. Diagnostic Applications of Antibodies Directed Against the Proteins of the Invention Antibodies directed against a protein of the invention may be used in methods known within the art relating to the localization and/or quantitation of the protein for use in measuring levels of the protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies against the proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antigen-binding domain, are utilized as pharmacologically-active compounds (see below).

An antibody specific for a protein of the invention can be used to isolate the protein by standard techniques, such as immunoaffinity chromatography or immunoprecipitation. Such an antibody can facilitate the purification of the natural protein antigen from cells and of recombinantly produced antigen expressed in host cells. Moreover, such an antibody can be used to detect the antigenic protein in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the antigenic protein. Antibodies directed against the protein can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, (3-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125, 131, 35S or H.

12. Antibody Therapeutics Antibodies of the invention, including polyclonal, monoclonal, humanized and fully human antibodies, may used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology in a subject. An antibody preparation, preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding witlhthe target. Such an effect may be one of two kinds, depending on the specific nature of the interaction between the given antibody molecule and the target antigen in question. In the first instance, administration of the antibody may abrogate or inhibit the binding of the target with an endogenous ligand to which it naturally binds. In this case, the antibody binds to the target and masks a binding site of the naturally WO 01/64884 PCT/US01/06839 occurring ligand, wherein the ligand serves as an effector molecule. Thus the receptor mediates a signal transduction pathway for which ligand is responsible.

Alternatively, the effect may be one in which the antibody elicits a physiological result by virtue of binding to an effector binding site on the target molecule. In this case the target, a receptor having an endogenous ligand that may be absent or defective in the disease or pathology, binds the antibody as a surrogate effector ligand, initiating a receptor-based signal transduction event by the receptor.

A therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target, and in other cases, promotes a physiological response. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Conmmon ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way ofnonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight.

Common dosing frequencies may range, for example, from twice daily to once a week.

13. Pharmaceutical Compositions of Antibodies Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhome, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

If the antigenic protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). The formulation herein WO 01/64884 PCT/US01/06839 can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.

Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration are highly preferred to be sterile.

This is readily accomplished by filtration through sterile filtration membranes.

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, films, or microcapsules.

Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly- D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acidglycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

gl2L Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a gl2L protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors non-episomal mammalian vectors) are WO 01/64884 PCT/US01/06839 integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form ofplasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein gl2L proteins, mutant forms of gl2L proteins, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed for expression of gl2L proteins in prokaryotic or eukaryotic cells. For example, gl2L proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.

WO 01/64884 PCT/US01/06839 (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, that fuse glutathione Stransferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 1 Id (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the gl2L expression vector is a yeast expression vector.

Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et al., 1987. EMBOJ. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif).

WO 01/64884 PCT/US01/06839 Alternatively, gl2L can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDMS (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187- 195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type tissuespecific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters the neurofilament promoter; Byme and Ruddle, 1989.

Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.

Science 230: 912-916), and mammary gland-specific promoters milk whey promoter; U.S.

Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentallyregulated promoters are also encompassed, the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989.

Genes Dev. 3: 537-546).

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to gl2L mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences WO 01/64884 PCT/US01/06839 can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, Weintraub, et al., "Antisense RNA as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, gl2L protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electropdration.

Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al.

(MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding gl2L or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid WO 01/64884 PCT/US01/06839 can be identified by drug selection cells that have incorporated the selectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce express) gl2L protein. Accordingly, the invention further provides methods for producing gl2L protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding gl2L protein has been introduced) in a suitable medium such that gl2L protein is produced. In another embodiment, the method further comprises isolating gl2L protein from the medium or the host cell.

Transgenic gl2L Animals The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which gl2L protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous gl2L sequences have been introduced into their genome or homologous recombinant animals in which endogenous gl2L sequences have been altered. Such animals are useful for studying the function and/or activity of gl2L protein and for identifying and/or evaluating modulators of gl2L protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gl2L gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, an embryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing gl2L-encoding nucleic acid into the male pronuclei of a fertilized oocyte by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human gl2L cDNA sequences of SEQ ID NO:1, can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue of the human gl2L gene, such as a mouse gl2L gene (SEQ ID NO:3), can be used as a transgene. Intronic WO 01/64884 PCT/US01/06839 sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably-linked to the gl2L transgene to direct expression of gl2L protein to particular cells.

Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the gl2L transgene in its genome and/or expression of gl2L mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding gl2L protein can further be bred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a gl2L gene into which a deletion, addition or substitution has been introduced to thereby alter, functionally disrupt, the gl2L gene. The gl2L gene can be a human gene the cDNA of SEQ ID NO: but more preferably, is a non-human homologue of a human gl2L gene. For example, a mouse homologue (SEQ ID NO:3) can be used to construct a homologous recombination vector suitable for altering an endogenous gl2L gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous gl2L gene is functionally disrupted no longer encodes a functional protein; also referred to as a "knock out" vector).

Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous gl2L gene is mutated or otherwise altered but still encodes functional protein the upstream regulatory region can be altered to thereby alter the expression of the endogenous gl2L protein). In the homologous recombination vector, the altered portion of the gl2L gene is flanked at its and 3'-termini by additional nucleic acid of the gl2L gene to allow for homologous recombination to occur between the exogenous gl2L gene carried by the vector and an endogenous gl2L gene in an embryonic stem cell. The additional flanking gl2L nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.

Typically, several kilobases of flanking DNA (both at the and 3'-termini) are included in the vector. See, Thomas, et al., 1987. Cell 51: 503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line by electroporation) and cells in which the introduced gl2L gene has homologously-recombined with the endogenous gl2L gene are selected. See, Li, et al., 1992. Cell 69: 915.

WO 01/64884 PCT/US01/06839 The selected cells are then injected into a blastocyst of an animal a mouse) to form aggregation chimeras. See, Bradley, 1987. In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechnol. 2: 823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.

In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, See, Lakso, et al., 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al., 1991. Science 251:1351-1355. Ifa cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilnut, et al., 1997. Nature 385: 810-813. In brief, a cell a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell the somatic cell) is isolated.

Pharmaceutical Compositions The gl2L nucleic acid molecules, gl2L proteins, and anti-gl2L antibodies (also referred to herein as "active compounds") of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for WO 01/64884 PCT/US01/06839 administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a phannaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Phannaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art.

Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral inhalation), transdennal topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and WO 01/64884 PCT/US01/06839 liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound a gl2L protein or anti-gl2L antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gun tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, a gas such as carbon dioxide, or a nebulizer.

WO 01/64884 PCT/US01/06839 Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.

Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdennal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, U.S. Patent No. 5,328,470) or by stereotactic injection (see, Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.

WO 01/64884 PCT/US01/06839 Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Screening and Detection Methods The isolated nucleic acid molecules of the invention can be used to express g12L protein via a recombinant expression vector in a host cell in gene therapy applications), to detect gl2L mRNA in a biological sample) or a genetic lesion in a gl2L gene, and to modulate gl2L activity, as described further, below. In addition, the gl2L proteins can be used to screen drugs or compounds that modulate the gl2L protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of gl2L protein or production of gl2L protein forms that have decreased or aberrant activity compared to gl2L wild-type protein metabolic disorders and diseases, such as obesity and obesity-related disorders, diabetes, and cachexia; cell proliferative disorders and diseases, such as hyperplasia, cancer, and restenosis; neurodegenerative disorders and diseases, such as Alzheimer's Disease and Parkinson's Disorder; immune disorders and diseases, such as AIDS, inflammation, and autoimmune diseases; and hematopoietic disorders and diseases, such as SCID, cyclic neutropenia, and thrombocythemia). In addition, the anti-gl2L antibodies of the invention can be used to detect and isolate gl2L proteins and modulate gl2L activity.

The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.

Screening Assays The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, candidate or test compounds or agents peptides, peptidomimetics, small molecules or other drugs) that bind to gl2L proteins or have a stimulatory or inhibitory effect on, gl2L protein expression or gl2L protein activity. The invention also includes compounds identified in the screening assays described herein.

In one embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate the activity of the membrane-bound form of a gl2L protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase WO 01/64884 PCT/US01/06839 libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997. Anticancer Drug Design 12: 145.

A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc.

Natl. Acad. Sci. U.S.A. 91: 11422; Zuckennann, et al., 1994. J. Med. Chem. 37: 2678; Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution Houghten, 1992.

Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S.

Patent 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).

In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of gl2L protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to an gl2L protein determined. The cell, for example, can of mammalian origin or a yeast cell.

Determining the ability of the test compound to bind to the gl2L protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the gl2L protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 125, 35S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or WO 01/64884 PCT/US01/06839 luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of gl2L protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds gl2L to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a gl2L protein, wherein determining the ability of the test compound to interact with a gl2L protein comprises determining the ability of the test compound to preferentially bind to gl2L protein or a biologically-active portion thereof as compared to the known compound.

In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of gl 2L protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate stimulate or inhibit) the activity of the gl2L protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of gl2L or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the gl2L protein to bind to or interact with a gl2L target molecule. As used herein, a "target molecule" is a molecule with which a gl2L protein binds or interacts in nature. A gl2L target molecule can be a non-gl2L molecule or a gl2L protein or polypeptide of the invention.

In one embodiment, a gl2L target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal a signal generated by binding of a compound to a membrane-bound gl2L molecule) through the cell membrane and into the cell.

The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with gl2L.

Determining the ability of the gl2L protein to bind to or interact with a gl2L target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the gl2L protein to bind to or interact with a gl2L target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target intracellular Ca 2 diacylglycerol, IP 3 etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a gl2L-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.

In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting a gl2L protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the gl2L protein or biologically-active WO 01/64884 PCT/US01/06839 portion thereof. Binding of the test compound to the gl2L protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the gl2L protein or biologically-active portion thereof with a known compound which binds gl2L to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a gl2L protein, wherein determining the ability of the test compound to interact with a gl2L protein comprises determining the ability of the test compound to preferentially bind to gl2L or biologically-active portion thereof as compared to the known compound.

In still another embodiment, an assay is a cell-free assay comprising contacting gl2L protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate stimulate or inhibit) the activity of the gl2L protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of gl2L can be accomplished, for example, by determining the ability of the gl2L protein to bind to a gl2L target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of gl2L protein can be accomplished by determining the ability of the gl2L protein further modulate a gl2L target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described, supra.

In yet another embodiment, the cell-free assay comprises contacting the gl2L protein or biologically-active portion thereof with a known compound which binds gl2L protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a gl2L protein, wherein determining the ability of the test compound to interact with a gl2L protein comprises determining the ability of the gl2L protein to preferentially bind to or modulate the activity of a gl2L target molecule.

The cell-free assays of the invention are amenable to use of both the soluble form or a membrane-bound form of gl2L protein. In the case of cell-free assays comprising the membrane-bound form of gl2L protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of gl2L protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton X- 100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, N-dodecyl--N,N-dimethyl- 3-ammonio-l-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate

(CHAPSO).

WO 01/64884 PCT/US01/06839 In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either gl2L protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to gl2L protein, or interaction of gl2L protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-gl 2L fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or gl2L protein, and the mixture is incubated under conditions conducive to complex formation at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of gl2L protein binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the gl2L protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated gl2L protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown within the art biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with g 2L protein or target molecules, but which do not interfere with binding of the gl2L protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or gl2L protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the gl2L protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the gl2L protein or target molecule.

In another embodiment, modulators of gl2L protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of gl2L mRNA or protein in the cell is determined. The level of expression of gl2L mRNA or protein in the presence of the candidate compound is compared to the level of expression of gl2L mRNA WO 01/64884 PCT/US01/06839 or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of gl2L mRNA or protein expression based upon this comparison. For example, when expression of g12L mRNA or protein is greater statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of gl2L mRNA or protein expression. Alternatively, when expression of gl2L mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of gl2L mRNA or protein expression. The level of gl2L mRNA or protein expression in the cells can be determined by methods described herein for detecting gl2L mRNA or protein.

In yet another aspect of the invention, the gl2L proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, U.S. Patent No. 5,283,317; Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chem. 268: 12046-12054; Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993. Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with gl2L ("gl2L-binding proteins" or "gl2L-bp") and modulate gl2L activity. Such gl2L-binding proteins are also likely to be involved in the propagation of signals by the gl2L proteins as, for example, upstream or downstream elements of the gl2L pathway.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for gl2L is fused to a gene encoding the DNA binding domain of a known transcription factor GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a gl2L-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein that interacts with gl2L.

The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.

WO 01/64884 PCT/US01/06839 Detection Assays Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) cat be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below.

Chromosome Mapping Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the g12L sequences, SEQ ID NOS:1 or 3, or fragments or derivatives thereof, can be used to map the location of the gl2L genes, respectively, on a chromosome. The mapping of the gl2L sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.

Briefly, gl2L genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the gl2L sequences. Computer analysis of the gl2L, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the gl2L sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from different mammals human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, D'Eustachio, et al., 1983. Science 220: 919-924.

Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a WO 01/64884 PCT/US01/06839 single thermal cycler. Using the g12L sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes.

Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.

Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (coinheritance of physically adjacent genes), described in, Egeland, et al., 1987. Nature, 325: 783-787.

Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the gl2L gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes WO 01/64884 PCT/US01/06839 from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

Tissue Typing The gl2L sequences of the invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences of the invention are useful as additional DNA markers for RFLP ("restriction fragment length polymorphisms," described in U.S. Patent No. 5,272,057).

Furthermore, the sequences of the invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the gl2L sequences described herein can be used to prepare two PCR primers from the and 3'-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The gl2L sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).

Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NOS:1 or 3, are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

Predictive Medicine The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining gl2L protein and/or WO 01/64884 PCT/US01/06839 nucleic acid expression as well as gl2L activity, in the context of a biological sample blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant gl2L expression or activity. The disorders include metabolic disorders and diseases, such as obesity and obesityrelated disorders, diabetes, and cachexia; cell proliferative disorders and diseases, such as hyperplasia, cancer, and restenosis; neurodegenerative disorders and diseases, such as Alzheimer's Disease and Parkinson's Disorder; immune disorders and diseases, such as AIDS, inflammation, and autoimmune diseases; and hematopoietic disorders and diseases, such as SCID, cyclic neutropenia, and thrombocythemia. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with gl2L protein, nucleic acid expression or activity. For example, mutations in a gl2L gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with gl2L protein, nucleic acid expression, or biological activity.

Another aspect of the invention provides methods for determining g12L protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics").

Pharmacogenomics allows for the selection of agents drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.) Yet another aspect of the invention pertains to monitoring the influence of agents drugs, compounds) on the expression or activity of gl2L in clinical trials.

These and other agents are described in further detail in the following sections.

Diagnostic Assays An exemplary method for detecting the presence or absence of gl2L in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting gl2L protein or nucleic acid mRNA, genomic DNA) that encodes gl2L protein such that the presence of gl2L is detected in the biological sample. An agent for detecting gl2L mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to gl2L mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length gl2L nucleic acid, such as the nucleic acid of SEQ ID NOS:1 or 3, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to gl2L mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.

WO 01/64884 PCT/US01/06839 An agent for detecting gl2L protein is an antibody capable of binding to gl2L protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof Fab or F(ab') 2 can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect gl2L mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of gl2L mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of gl2L protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of gl2L genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of gl2L protein include introducing into a subject a labeled anti-gl2L antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is an adipose tissue sample, a tumor biobsy, or a blood sample.

In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting gl2L protein, mRNA, or genomic DNA, such that the presence of gl2L protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of gl2L protein, mRNA or genomic DNA in the control sample with the presence of gl2L protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of gl2L in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting gl2L protein or mRNA in a biological sample; means for determining the amount of gl2L in the sample; and means for comparing the amount of gl2L in the sample with a standard. The WO 01/64884 PCT/US01/06839 compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect gl2L protein or nucleic acid.

Prognostic Assays The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant g12L expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with gl2L protein, nucleic acid expression or activity. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with aberrant gl2L expression or activity in which a test sample is obtained from a subject and g12L protein or nucleic acid mRNA, genomic DNA) is detected, wherein the presence of gl2L protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant gl2L expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant gl2L expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder. Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant gl2L expression or activity in which a test sample is obtained and gl2L protein or nucleic acid is detected wherein the presence of gl2L protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant gl2L expression or activity).

The methods of the invention can also be used to detect genetic lesions in a gl2L gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding a g12Lprotein, or the misexpression of the gl2L gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: a deletion of one or more nucleotides from a gl2L gene; (ii) an addition of one or more nucleotides to a gl2L gene; (iii) a substitution of one or more nucleotides of a gl2L gene, (iv) a chromosomal rearrangement of a gl2L gene; WO 01/64884 PCT/US01/06839 an alteration in the level of a messenger RNA transcript of a gl2L gene, (vi) aberrant modification of a gl2L gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of a gl2L gene, (viii) a non-wild-type level of a gl2L protein, (ix) allelic loss of a gl2L gene, and (x) inappropriate post-translational modification of a gl2L protein. As described herein, there are a large number of assay techniques known in the art that can be used for detecting lesions in a gl2L gene. A preferred biological sample is an adipose tissue sample, tumor biopsy, or a blood sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used.

In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and Nakazawa, et al., 1994. Proc. Natl. Acad.

Sci. USA 91: 360-364), the latter of which can be particularly useful for detecting point mutations in the gl2L-gene (see, Abravaya, et al., 1995. Nucl. Acids Res. 23: 675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to a gl2L gene under conditions such that hybridization and amplification of the gl2L gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); QP Replicase (see, Lizardi, et al, 1988. BioTechnology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

In an alternative embodiment, mutations in a gl2L gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared.

Differences in fragment length sizes between sample and control DNA indicates mutations in WO 01/64884 PCT/US01/06839 the sample DNA. Moreover, the use of sequence specific ribozymes (see, U.S. Patent No.

5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in gl2L can be identified by hybridizing a sample and control nucleic acids, DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes. See, Cronin, et al., 1996. Human Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example, genetic mutations in gl2L can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the gl2L gene and detect mutations by comparing the sequence of the sample gl2L-with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl.

Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, Naeve, et al., 1995. Biotechniques 19: 448), including sequencing by mass spectrometry (see, PCT International Publication No. WO 94/16101; Cohen, et al., 1996. Adv. Chromatography 36: 127-162; and Griffin, et al., 1993.

Appl. Biochem. Biotechnol. 38: 147-159).

Other methods for detecting mutations in the gl2L gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See, Myers, et al., 1985. Science 230: 1242. In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type gl2L sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance; RNA/DNA duplexes can be treated with RNase and DNAIDNA hybrids treated with Si nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can WO 01/64884 PCT/US01/06839 be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al., 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, et al., 1992. Methods Enzymol. 217: 286-295. In an embodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in gl2L cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches. See, Hsu, et al., 1994. Carcinogenesis 15: 1657-1662. According to an exemplary embodiment, a probe based on a gl2L sequence, a wild-type gl2L sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, U.S. Patent No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in gl2L genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, Orita, et al., 1989. Proc. Natl. Acad. Sci. USA: 86: 2766; Cotton, 1993. Mutat.

Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79. Single-stranded DNA fragments of sample and control gl2L nucleic acids will be denatured and allowed to renature.

The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, Keen, et al., 1991. Trends Genet. 7: In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE). See, Myers, et al., 1985. Nature 313: 495. When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient WO 01/64884 PCT/US01/06839 to identify differences in the mobility of control and sample DNA. See, Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension.

For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, Saiki, et al., 1986. Nature 324: 163; Saiki, et al., 1989. Proc.

Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; see, Gibbs, et al., 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech. 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection. See, Gasparini, et al., 1992. Mol. Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, Barany, 1991. Proc. Natl. Acad.

Sci. USA 88: 189. In such cases, ligation will occur only if there is a perfect match at the 3'terminus of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a gl2L gene.

Furthermore, any cell type or tissue in which gl2L is expressed may be utilized in the prognostic assays described herein.

Pharmacogenomics Agents, or modulators that have a stimulatory or inhibitory effect on gl2L activity gl2L gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (The disorders include metabolic disorders and diseases, such as obesity and obesity-related disorders, diabetes, and cachexia; cell proliferative disorders and diseases, such as hyperplasia, cancer, and restenosis; WO 01/64884 PCT/US01/06839 neurodegenerative disorders and diseases, such as Alzheimer's Disease and Parkinson's Disorder; immune disorders and diseases, such as AIDS, inflammation, and autoimmune diseases; and hematopoietic disorders and diseases, such as SCID, cyclic neutropenia, and thrombocythemia.) In conjunction with such treatment, the phannacogenomics the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype.

Such phannacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of gl2L protein, expression of gl2L nucleic acid, or mutation content of gl2L genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.

Phannacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985; Linder, 1997. Clin. Chem., 43: 254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of WO 01/64884 PCT/US01/06839 CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-fonned metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

Thus, the activity of gl2L protein, expression of gl2L nucleic acid, or mutation content of g12L genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. Tn addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a gl2L modulator, such as a modulator identified by one of the exemplary screening assays described herein.

Monitoring of Effects During Clinical Trials Monitoring the influence of agents drugs, compounds) on the expression or activity of gl2L the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase g12L gene expression, protein levels, or upregulate gl2L activity, can be monitored in clinical trails of subjects exhibiting decreased gl2L gene expression, protein levels, or downregulated g12L activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease gl2L gene expression, protein levels, or downregulate gl2L activity, can be monitored in clinical trails of subjects exhibiting increased gl2L gene expression, protein levels, or upregulated gl2L activity. In such clinical trials, the expression or activity of gl2L and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers of the immune responsiveness of a particular cell.

By way of example, and not of limitation, genes, including gl2L, that are modulated in cells by treatment with an agent compound, drug or small molecule) modulating g12L activity identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of gl2L and other genes implicated in the disorder. The levels of gene expression a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by WO 01/64884 PCT/US01/06839 measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of gl2L or other genes. In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent.

Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.

In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a gl2L protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the gl2L protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the gl2L protein, mRNA, or genomic DNA in the pre-administration sample with the gl2L protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of gl2L to higher levels than detected, to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of gl2L to lower levels than detected, to decrease the effectiveness of the agent.

Methods of Treatment The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant gl2L expression or activity. The disorders include metabolic disorders and diseases, such as obesity and obesity-related disorders, diabetes, and cachexia; cell proliferative disorders and diseases, such as hyperplasia, cancer, and restenosis; neurodegenerative disorders and diseases, such as Alzheimer's Disease and Parkinson's Disorder; inunune disorders and diseases, such as AIDS, inflammation, and autoimmune diseases; and hematopoietic disorders and diseases, such as SCID, cyclic neutropenia, and thrombocythemia. These methods of treatment will be discussed more fully, below.

Disease and Disorders Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with WO 01/64884 PCT/US01/06839 Therapeutics that antagonize reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to: an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endogenous function of an aforementioned peptide by homologous recombination (see, Capecchi, 1989.

Science 244: 1288-1292); or modulators inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between an aforementioned peptide and its binding partner.

Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.

Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression ofmRNAs Northern assays, dot blots, in situ hybridization, and the like).

Prophylactic Methods In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant gl2L expression or activity, by administering to the subject an agent that modulates gl2L expression or at least one gl2L activity. Subjects at risk for a disease that is caused or contributed to by aberrant gl2L expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the gl2L aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of gl2L aberrancy, for example, a gl2L agonist or gl2L antagonist agent can be used for treating the subject. The WO 01/64884 PCT/US01/06839 appropriate agent can be determined based on screening assays described herein. The prophylactic methods of the invention are further discussed in the following subsections.

Therapeutic Methods Another aspect of the invention pertains to methods of modulating gl2L expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of g12L protein activity associated with the cell. An agent that modulates gl2L protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a gl2L protein, a peptide, a gl2L peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more g12L protein activity. Examples of such stimulatory agents include active g12L protein and a nucleic acid molecule encoding g12L that has been introduced into the cell. In another embodiment, the agent inhibits one or more gl2L protein activity.

Examples of such inhibitory agents include antisense gl2L nucleic acid molecules and antigl2L antibodies. These modulatory methods can be performed in vitro by culturing the cell with the agent) or, alternatively, in vivo by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a gl2L protein or nucleic acid molecule. In one embodiment, the method involves administering an agent an agent identified by a screening assay described herein), or combination of agents that modulates up-regulates or down-regulates) g12L expression or activity. In another embodiment, the method involves administering a g12L protein or nucleic acid molecule as therapy to compensate for reduced or aberrant g12L expression or activity.

Stimulation of gl2L activity is desirable in situations in which g12L is abnormally downregulated and/or in which increased gl2L activity is likely to have a beneficial effect. One example of such a situation is where a subject has a disorder characterized by aberrant cell proliferation and/or differentiation cancer or immune associated disorders). Another example of such a situation is where the subject has a gestational disease preclampsia).

Determination of the Biological Effect of the Therapeutic In various embodiments of the invention, suitable in vitro or in vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue.

In various specific embodiments, in vitro assays may be performed with representative cells of the type(s) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s). Compounds for use in therapy may be tested in suitable WO 01/64884 PCT/US01/06839 animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model system known in the art may be used prior to administration to human subjects.

Prophylactic and Therapeutic Uses of the Compositions of the Invention The gl2L nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to: metabolic disorders and diseases, such as obesity and obesity-related disorders, diabetes, and cachexia; cell proliferative disorders and diseases, such as hyperplasia, cancer, and restenosis; neurodegenerative disorders and diseases, such as Alzheimer's Disease and Parkinson's Disorder; immune disorders and diseases, such as AIDS, inflammation, and autoimmune diseases; and hematopoietic disorders and diseases, such as SCID, cyclic neutropenia, and thrombocythemia.

As an example, a cDNA encoding the gl2L protein of the invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the invention will have efficacy for treatment of patients suffering from metabolic disorders and diseases, such as obesity and obesity-related disorders, diabetes, and cachexia; cell proliferative disorders and diseases, such as hyperplasia, cancer, and restenosis; neurodegenerative disorders and diseases, such as Alzheimer's Disease and Parkinson's Disorder; immune disorders and diseases, such as AIDS, inflammation, and autoimmune diseases; and hematopoietic disorders and diseases, such as SCID, cyclic neutropenia, and thrombocytheinia.

Both the novel nucleic acid encoding the gl2L protein, and the gl2L protein of the invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. A further use could be as an anti-bacterial molecule some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of antibodies that immunospecifically-bind to the novel substances of the invention for use in therapeutic or diagnostic methods.

EXAMPLES

The following examples illustrate by way of non-limiting example various aspects of the invention.

As is the case for Spot 14, which is differentially regulated during BAT differentiation, mouse gl2L is also modulated in BAT under conditions that affect the metabolic activity and proliferative status of BAT. Under the experimental conditions for the identification of WO 01/64884 PCT/US01/06839 differentially expressed genes in BAT, a fragment of cDNA (representing an mRNA encoding mouse gl2L) was modulated in response to cold The pattern of modulation observed for the cDNA fragment in BAT from animals raised under the experimental conditions described above was up-regulation in cold-acclimated vs. control, no change in warm-acclimated vs.

control, and up-regulation in cold-acclimated vs. warm-acclimated. Over time mouse gl2L cDNA fragment is up-regulated, and remains up-regulated, under the condition of coldacclimation. The TaqMan-derived tissue distribution of mouse gl2L shows that the mouse proteins expression is significantly elevated in the response to the cold in WAT and BAT relative to the same tissues from warm-acclimated animals.

The identification of the modulated fragment lead to the identification of a full length mouse cDNA (Table 2A; SEQ ID NO:3) encoding a 182 amino acid protein (Table 2B; SEQ ID NO:4) of predicted molecular weight 20355.6 D, predicted pi 5.33, and predicted physical characteristics in Table 3.

A human protein homolog (Table 1B; SEQ ID NO:2) was also identified, 183 amino acids, 20233.5 D, predicted pl=5.55, and other predicted physical characteristics (Table An optimal cDNA was determined by assembly of 85 ESTs from public and private databases (Table 1A; SEQ ID NO:1). The pi's of the mouse, human homologs of the zebrafish g12 protein, and the zebrafish gl2 (predicted pi 4.93) proteins have more alkaline predicted pi's than the Spot 14 family proteins (Rat Spot 14 pi 4.61, Mouse Spot 14 pi 4.76, Human Spot 14 pi 4.64) due to the more extensive acid-rich domains of the Spot 14's (Grillasca, FEBS Lett. 1997 Jan 13;401(1):38-42) compared to the g12 family members. This and other data support the contention that the novel mouse and human gl2L molecules described herein are the true homologs of the zebrafish gastrulation-specific protein, gl2.

Sequence analysis indicates that the g12 family members have a "spot box" and an "acid box" observed for members of the Spot 14 family members (FIG. The g12 family members have a conserved insert just after the spot box (the zebrafish insert is significantly smaller than the inserts for the mouse and human gl2L's) and a second highly conserved insert just after amino acid 54 of the mouse sequence. The g12 family members have a somewhat less acidic acid box (amino acids 87-114 of the mouse Spot 14 polypeptide) as compared to the Spot 14 family members (FIG. This reduced acid box likely accounts for the relatively higher pi's of the g12 family members compared to the Spot 14 family members.

Mouse and human gl2L are more similar to zebrafish g12 than the Spot 14's, both at the protein level and at the level of the nucleotide sequence.

WO 01/64884 PCTIUSO1/06839 The mouse gl2L maps to cliromosome X at position DXMit89 in mouse by radiation hybrid mapping. The human gl2L gene is found on chromosome X and is 100% identical to GeneBank AK001428 (Submitted (1 6-FEB-2000) to the DDBS/EMBL/Genlank).

WO 01/64884 PCT/US01/06839 Table 3 Predicted physical characteristics of Mouse gl2L protein Values assuming ALL Cys residues appear as half cystines: Values assuming NO Cys residues appear as half cystines: Note: The conditions at which these equations are valid are: pH 6.5, 6.0 M guanidium hydrochloride, 0.02 M phosphate buffer.

WO 01/64884 PCT/US01/06839 Table 4 Predicted physical characteristics of Human gl2L protein.

Values assuming ALL Cys residues appear as half cystines: 276 nm 278 nm 279 nm 280 nm 282 nm SExtinction Coefficient 23595 23927 23825 23590 22900 Optical Density 1.166 1.183 1.178 1.166 1.132 Values assuming NO Cys residues appear as half cystines: 276 nm 278 nm 279 nm 280 nm 282 nm Extinction Coefficient 23450 23800 23705 23470 1 22800 Optical Density 1.159 1.176 1.172 1.160 1.127 Note: The conditions at which these equations are valid are: pH 6.5, 6.0 M guanidium hydrochloride, 0.02 Mphospha buffer.

In summary, the inventors have identifying novel mouse and human genes responsive to the thermal state of animals raised below their thermal neutral zone as a means of identifying such genes modulated in response to the metabolic status and responsive to thermal conditions.

It is contemplated that such molecules relate to the endocrine nature of BAT and its role in, and responsiveness to, thermoregulation and metabolism.

EQUIVALENTS

Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims that follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein. Other aspects, advantages, and modifications considered to be within the scope of the following claims.

Claims (55)

1. 2. A polypeptide according to claim 1, wherein said polypeptide comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid comprising SEQ ID NO: 2.
2. 3. A polypeptide according to claim 2, wherein said allelic variant comprises an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from a nucleic acid comprising SEQ ID NO: 1.
3. 4. A polypeptide according to any one of claims 1 to 3, wherein the amino acid sequence of said variant comprises a conservative amino acid substitution. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: a mature form of an amino acid sequence comprising SEQ ID NO: 4; a variant of a mature form of an amino acid sequence comprising SEQ ID NO: 4, wherein one or more amino acid residues in said variant differs from H:\terryr\Keep\Retype\P46923 Amended Claims Curagen Genentech- July 2005.doc 13/07/05 86 the amino acid sequence of said mature form, provided that said variant differs in no more than 10% of the amino acid residues from the amino acid sequence of said mature form; an amino acid sequence comprising SEQ ID NO: 4; and a variant of an amino acid comprising SEQ ID NO: 4, wherein one or more amino acid residues in said variant differs from the amino acid sequence of SEQ ID NO: 4, provided that said variant differs in no more than 10% of amino acid residues from said amino acid sequence; wherein the expression of the polypeptide is upregulated in white adipose tissue or brown adipose tissue in response to cold.
4. 6. A polypeptide according to claim 5, wherein said polypeptide comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid comprising SEQ ID NO: 4.
5. 7. A polypeptide according to claim 6, wherein said allelic variant comprises an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from a nucleic acid comprising SEQ ID NO: 3.
6. 8. A polypeptide according to any one of claims 5 to 7, wherein the amino acid sequence of said variant comprises a conservative amino acid substitution.
7. 9. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: a mature form of an amino acid sequence comprising SEQ ID NO: 2; a variant of a mature form of an amino acid sequence comprising SEQ ID NO: 2, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 3% of the amino acid residues from the amino acid sequence of said mature form; an amino acid sequence comprising SEQ ID NO: 2; a variant of an amino acid comprising SEQ ID NO: 2, wherein one or more amino acid residues in said variant differs from the amino acid sequence of H:\terryr\Keep\Retype\P46923 Amended Claims Curagen Genentech- July 2005.doc 13/07/05 87 Ssaid mature form, provided that said variant differs in no more than 3% of amino acid residues from said amino acid sequence; and a nucleic acid molecule comprising the complement of m or wherein the transcription of the nucleic acid is upregulated in white adipose tissue or brown adipose tissue in response to cold. A nucleic acid molecule according to claim 9, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally-occurring allelic nucleic acid Svariant. (N
8. 11. A nucleic acid molecule according to claim 9 or claim 10, wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of a naturally-occurring polypeptide variant.
9. 12. A nucleic acid molecule according to any one of claims 9 to 11, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid comprising SEQ ID NO: 1.
10. 13. A nucleic acid molecule according to claim 9, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a nucleotide sequence comprising SEQ ID NO: 1; a nucleotide sequence differing by one or more nucleotides from a nucleotide sequence comprising SEQ ID NO: 1, provided that no more than 20% of the nucleotides differ from said nucleotide sequence.
11. 14. A nucleic acid molecule according to any one of claims 9 to 13, wherein said nucleic acid molecule hybridizes under stringent conditions to a nucleotide sequence comprising SEQ ID NO: 1, or a complement of said nucleotide sequence. A nucleic acid molecule according to claim 9, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a first nucleotide sequence comprising a coding sequence differing by one or more nucleotide sequences from a coding sequence encoding said amino acid sequence, provided that no more than 20% of the nucleotides in the coding sequence in said first nucleotide sequence differ from said coding sequence; and H:\terryr\Keep\Retype\P46923 Amended Claims Curagen Genentech- July 2005.doc 13/07/05 88 an isolated second polynucleotide that is a complement of the first polynucleotide. C 16. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: a mature form of an amino acid sequence comprising SEQ ID SNO: 4; C a variant of a mature form of an amino acid sequence comprising SEQ ID NO: 4, wherein one or more amino acid residues in said variant differs from O the amino acid sequence of said mature form, provided that said variant differs in no more than 10% of the amino acid residues from the amino acid sequence of said mature form; an amino acid sequence comprising SEQ ID NO: 4; a variant of an amino acid comprising SEQ ID NO: 4, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 10% of amino acid residues from said amino acid sequence; and a nucleic acid molecule comprising the complement of or wherein the transcription of the nucleic acid is upregulated in white adipose tissue or brown adipose tissue in response to cold.
12. 17. A nucleic acid molecule according to claim 16, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally-occurring allelic nucleic acid variant.
13. 18. A nucleic acid molecule according to claim 16, wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of a naturally- occurring polypeptide variant.
14. 19. A nucleic acid molecule according to any one of claims 16 to 18, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid comprising SEQ ID NO: 3. H:\terryr\Keep\Retype\P46923 Amended Claims Curagen Genentech- July 2005.doc 13/07/05 89 A nucleic acid molecule according to claim 16, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: S(a) a nucleotide sequence comprising SEQ ID NO: 3; C a nucleotide sequence differing by one or more nucleotides from a nucleotide sequence comprising SEQ ID NO: 3, provided that no more than 20% of the nucleotides differ from said nucleotide sequence.
15. 21. A nucleic acid molecule according to claim 16, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a first nucleotide sequence comprising a coding sequence O differing by one or more nucleotide sequences from a coding sequence encoding said amino acid sequence, provided that no more than 20% of the nucleotides in the coding sequence in said first nucleotide sequence differ from said coding sequence; and an isolated second polynucleotide that is a complement of the first polynucleotide.
16. 22. A vector comprising a nucleic acid molecule according to any one of claims 9 to 21.
17. 23. A vector according to claim 22, further comprising a promoter operably- linked to said nucleic acid molecule.
18. 24. A cell comprising a vector according to claim 22 or claim 23.
19. 25. An isolated antibody that binds immunospecifically to a polypeptide according to any one of claims 1 to 8.
20. 26. An antibody according to claim 25, wherein said antibody is a monoclonal antibody.
21. 27. An antibody according to claim 25, wherein the antibody is a humanized antibody.
22. 28. A method for determining the presence or amount of a polypeptide according to any one of claims 1 to 8 in a sample, the method comprising: providing the sample; H:\terryr\Keep\Retype\P46923 Amended Claims Curagen Genentech- July 2005.doc 13/07/05 90 contacting the sample with an antibody that binds immunospecifically to the polypeptide; and S(c) determining the presence or amount of antibody bound to said c polypeptide, thereby determining the presence or amount of polypeptide in said sample.
23. 29. A method for determining the presence or amount of a nucleic acid molecule according to any one of claims 9 to 21 in a sample, the method comprising: t providing the sample; contacting the sample with a probe that binds to said nucleic acid molecule; and determining the presence or amount of the probe bound to said nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in said sample.
24. 30. A method according to claim 29, wherein presence or amount of the nucleic acid molecule is used as a marker for cell or tissue type.
25. 31. A method according to claim 30, wherein the cell or tissue type is cancerous.
26. 32. A method of identifying an agent that binds to a polypeptide according to any one of claims 1 to 8, the method comprising: contacting said polypeptide with said agent; and determining whether said agent binds to said polypeptide.
27. 33. A method according to claim 32, wherein the agent is a cellular receptor or a downstream effector.
28. 34. A method for identifying an agent that modulates the expression or activity of a polypeptide according to any one claims 1 to 8, the method comprising: providing a cell expressing said polypeptide; contacting the cell with said agent, and determining whether the agent modulates expression or activity of said polypeptide, whereby an alteration in expression or activity of said peptide indicates said agent modulates expression or activity of said polypeptide. H:\terryr\Keep\Retype\P46923 Amended Claims Curagen Genentech- July 2005.doc 13/07/05 91 A method for modulating the activity of a polypeptide according to any one of claims 1 to 8, the method comprising contacting a cell sample expressing the polypeptide of said claim with a compound that binds to said polypeptide in an amount c sufficient to modulate the activity of the polypeptide.
29. 36. A method of treating or preventing a gl2L-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired a Ipolypeptide according to any one of claims 1 to 8, in an amount sufficient to treat or prevent said gl2L-associated disorder in said subject.
30. 37. A method according to claim 36, wherein the disorder is selected from the group consisting of cancer, obesity, obesity-related disorders, diabetes, and cachexia.
31. 38. A method according to claim 36 or claim 37, wherein the disorder is related to metabolism.
32. 39. A method according to any one of claims 36 to 38, wherein said subject is a human.
33. 40. A method of treating or preventing a gl2L-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired a nucleic acid according to any one of claims 9 to 21, in an amount sufficient to treat or prevent said gl2L-associated disorder in said subject.
34. 41. A method according to claim 40, wherein the disorder is selected from the group consisting of cancer, obesity, obesity-related disorders, diabetes, and cachexia.
35. 42. A method according to claim 40 or claim 41, wherein the disorder is related metabolism.
36. 43. A method according to any one of claims 40 to 42, wherein said subject is a human.
37. 44. A method of treating or preventing a gl2L-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired an antibody according to any one of claims 25 to 27, in an amount sufficient to treat or prevent said gl2L-associated disorder in said subject. H:\terryr\Keep\Retype\P46923 Amended Claims Curagen Genentech- July 2005.doc 13/07/05 92 A method according to claim 44, wherein the disorder is selected from the Zgroup consisting of cancer, obesity, obesity-related disorders, diabetes, and cachexia.
38. 46. A method according to claim 44 or claim 45, wherein the disorder is related to metabolism. S47. A method according to any one of claims 44 to 46, wherein the subject is a human.
39. 48. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 8 and a pharmaceutically-acceptable carrier.
40. 49. A pharmaceutical composition comprising a nucleic acid molecule according to any one of claims 9 to 21 and a pharmaceutically-acceptable carrier. A pharmaceutical composition comprising an antibody according to any one of claims 25 to 27 and a pharmaceutically-acceptable carrier.
41. 51. A kit comprising in one or more containers, a pharmaceutical composition according to any one of claims 48 to
42. 52. A method for determining the presence of or predisposition to a disease associated with altered levels of a polypeptide according to any one of claims 1 to 8 in a first mammalian subject, the method comprising: measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and comparing the amount of said polypeptide in the sample of step to the amount of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, said disease; wherein an alteration in the expression level of the polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to said disease.
43. 53. A method according to claim 52, wherein the predisposition is to cancers. H:\terryr\Keep\Retype\P46923 Amended Claims Curagen Genentech- July 2005.doc 13/07/05 93
44. 54. A method for determining the presence of or predisposition to a disease N associated with altered levels of a nucleic acid molecule according to any one of claims Z9 to 21 in a first mammalian subject, the method comprising: c measuring the amount of the nucleic acid in a sample from the first mammalian subject; and comparing the amount of said nucleic acid in the sample of step to the amount of the nucleic acid present in a control sample from a second t mammalian subject known not to have or not be predisposed to, the disease; wherein an alteration in the level of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease. A method according to claim 54, wherein the predisposition is to cancers.
45. 56. A method of treating a pathological state in a mammal, the method comprising administering to a mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 97% identical to a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 and expression of the polypeptide is upregulated in white adipose tissue or brown adipose tissue in response to cold.
46. 57. A method of treating a pathological state in a mammal, the method comprising administering to the mammal an antibody according to any one of claims to 27 in an amount sufficient to alleviate the pathological state.
47. 58. A method of measuring g l 2L transcription up-regulation or down- regulation activity of a compound, comprising: contacting said compound with a composition comprising a RNA polymerase and a polynucleotide according to any one of claims 9 to 21.
48. 59. A method according to claim 58, wherein said composition is in a cell. A method of measuring gl 2L translation up-regulation or down-regulation activity of a compound, comprising: contacting said compound with a composition comprising a ribosome and a polynucleotide according to any one of claims 9 to 21.
49. 61. A method according to claim 60, wherein said composition is in a cell. H:\terryr\Keep\Retype\P46923 Amended Claims Curagen Genentech- July 2005.doc 13/07/05 94 O O
50. 62. Use of a nucleic acid according to any one of claims 9 to 21 in the manufacture of a medicament for the treatment of a gl2L-associated disorder.
51. 63. Use of an antibody according to any one of claims 25 to 27 in the manufacture of a medicament for the treatment of a gl 2L-associated disorder.
52. 64. Use of a polypeptide according to any one of claims 1 to 8 in the manufacture of a medicament for the treatment of a pathological state in a mammal. Use of an antibody according to any one of claims 25 to 27 in the manufacture of a medicament for the treatment of a pathological state.
53. 66. An isolated polypeptide according to claim 1 substantially as hereinbefore described with reference to any one of the Examples.
54. 67. An isolated nucleic acid according to claim 9 substantially as hereinbefore described with reference to any one of the Examples.
55. 68. An method according to claim 28 substantially as hereinbefore described with reference to any one of the Examples. Dated this 13th day of July 2005 CURAGEN CORPORATION and GENENTECH, INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\terryr\Keep\Retype\P46923 Amended Claims Curagen Genentech- July 2005.doc 13/07/05