Definitions

• Sequence Listing is provided as an electronic file (PJZ04_seqList.txt, 930,050 bytes in size, created on January 13, 2001) on four identical compact discs (CD-R), labeled "COPY 1," "COPY 2,” “COPY 3,” and "CRF.”
• CD-R compact discs
• the Sequence Listing complies with Annex C of the Administrative Instructions, and may be viewed, for example, on an IBM-PC machine running the MS-Windows operating system by using the V viewer software, version 2000 (see World Wide Web URL: http://www.fileviewer.com).
• the present invention relates to novel proteins. More specifically, isolated nucleic acid molecules are provided encoding novel polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing, treating, preventing and/or prognosing disorders related to these novel polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further relates to methods and/or compositions for inhibiting or enhancing the production and function ofthe polypeptides ofthe present invention.
• the human endocrine system is a collection of specialized organs and body tissues that produce, store, and secrete hormones. Endocrine organs are important in regulating the body's growth and development, controlling the function of various tissues, supporting pregnancy and other reproductive functions, and regulating metabolism. [5] Endocrine organs are sometimes called ductless glands because they have no ducts connecting them to specific body parts. The hormones they secrete are released into the bloodstream.
• the primary glands that make up the human endocrine system are the hypothalamus, the pituitary gland, the thyroid gland, the parathyroid glands, the islets of the pancreas (which produce insulin), the adrenal glands, the ovaries and the testes.
• the placenta also acts as an endocrine gland in addition to its other functions. Hormones secreted by these glands control physical growth, sexual function, metabolism, and other functions.
• hormones are steroids, fatty substances derived from cholesterol.
• Very small amounts of hormones can trigger very large responses in the body.
• Hormones bind to receptors on a cell's surface or inside a cell. The binding of a hormone to a receptor speeds up, slows down, or in some other way alters the cell's function.
• hormones control the function of entire organs. They control growth and development, reproduction, and sexual characteristics. They influence the way the body uses and stores energy. Hormones also control the volume of fluid and the levels of salt and sugar in the blood. Some hormones affect only one or two organs, while others affect the whole body. For example, thyroid-stimulating hormone is produced in the pituitary gland, and affects only the thyroid gland.
• Endocrine system or hormone imbalance disorders can be classified as disturbances of hormone production, or insensitivities of target tissues to hormonal effects.
• the first type, called production disorders are " divided into hypofunction disorders (insufficient activity) and hyperfunction disorders (excess activity).
• Hypofunction disorders can have a variety of causes, including malformations in the endocrine gland itself. More commonly, hypofunction is caused by disease or injury. Hypofunction can also result, when target tissue is unable to respond to hormones.
• Hyperfunction can be caused by glandular tumors that secrete hormone without responding to feedback controls.
• some autoimmune conditions create antibodies that have the side effect of stimulating hormone production.
• Infection of an endocrine gland can have the same result.
• Many of the endocrine disorders that affect adults also affect children but may produce different symptoms.
• the pituitary gland a pea-sized gland at the base of the brain, produces a number of hormones, such as corticotropin, thyroid-stimulating hormone, follicle-stimulating hormone, and luteinizing hormone, which control the function of other endocrine glands and stimulate these glands to produce other hormones.
• Luteinizing hormone LH
• follicle-stimulating hormone follitropin
• FSH chorionic gonadotropin
• gonadotropic hormones because of their actions on gonadal cells.
• the primary physiological effect of the gonadotropins is the promotion of gametogenesis and/or gonadal steroid production.
• Quantitative analyses of plasma LH and FSH levels as determined by radioimmunoassays and/or bioassays specific for each hormone, are useful in the diagnoses of several reproductive disorders. In both males and females, low or undetectable levels of LH and FSH are indicative of hypogonadotropic hypogonadism and suggest hypothalamic disease or pituitary failure.
• hypopituitarism may be caused by a noncancerous pituitary tumor (craniopharyngioma), an injury, or an infection, or it may have no identifiable cause (idiopathic hypopituitarism). Rarely, hypopituitarism occurs as part of Hand-Schuller-Christian disease, which affects small areas of bone and lungs and the function ofthe pituitary gland.
• This disorder can result when the entire pituitary gland is damaged.
• testes develop and produce sperm, because these functions are controlled by follicle-stimulating hormone, but the testes do not produce enough testosterone.
• Testosterone is the hormone that stimulates the development of male secondary sexual characteristics, such . as deepening of the voice, growth of facial hair, and maturation of the penis. Consequently, boys who have isolated luteinizing hormone deficiency do not develop these characteristics. Abnormally long arms and legs may be another symptom of this deficiency.
• thyroid gland produces thyroid hormone, which controls the speed at which the body's chemical functions proceed (metabolic rate).
• Thyroid hormones are the best known iodine-containing compounds with biological function. They have two functions in the human: as a crucial determinant of normal development (especially of the central nervous system) and in adults, to maintain metabolic homeostasis. Thyroid disorders are common with two general presentations: changes in the shape and/or size of the gland, or changes in secretion of hormones from the gland.
• Thyroid nodules and goiter the most common endocrinopathies, can be caused by benign or malignant tumors.
• a goiter may exist if the thyroid gland is underactive (producing too little thyroid hormone) or overactive (producing too much).
• An enlarged thyroid gland present at birth is called congenital goiter.
• Some children have Pendred's syndrome, a hereditary condition that combines deaf-mutism and congenital goiter.
• Hypothyroidism, or myxedema when severe results when the thyroid gland cannot produce adequate amounts of thyroid hormone for the body's needs. Worldwide, hypothyroidism is most often the result of iodine deficiency.
• hypothyroidism In nonendemic areas where iodine is sufficient, chronic autoimmune thyroiditis (Hashimoto's thyroiditis) accounts for the majority of cases. Hypothyroidism present at birth is known as congenital hypothyroidism and is the most common .preventable cause of mental retardation in the world. Diagnosis and early intervention with thyroid hormone replacement prevent the development of cretinism. Hypothyroidism that begins in childhood (juvenile hypothyroidism) slows growth, sometimes resulting in disproportionately short limbs. Tooth development is delayed. Hypothyroidism. that begins in adolescence (adolescent hypothyroidism) is similar to that in adults, except that it may delay puberty. Symptoms include a hoarse voice, slowed speech, droopy eyelids, a puffy face, hair loss, dry skin, a slow pulse, and weight gain.
• Hyperthyroidism results from overactivity of the thyroid gland. In newborns, the most common cause of hyperthyroidism is neonatal Graves' disease, a potentially life- threatening illness that can occur in infants whose mothers have or have had Graves' disease. Graves' disease, a form of hyperthyroidism, is an autoimmune disorder in which the body . produces antibodies that stimulate the thyroid gland. In pregnant women, these antibodies cross the placenta and stimulate the fetus' thyroid gland. Graves' disease in the mother may result in stillbirth, miscarriage, or premature birth.
• Islet cells of the pancreas synthesize and secrete insulin, the primary hormone responsible for controlling the uptake, utilization, arid storage of cellular nutrients.
• diabetes mellitus One ofthe most common diseases of the endocrine system is diabetes mellitus. Most patients can be classified as having either insulin-dependent diabetes mellitus (type I diabetes) or non- insulin-dependent diabetes. mellitus (type II diabetes). Virtually all forms of diabetes mellitus are due to a decrease in the circulating concentration of insulin and a decrease in the response of peripheral tissues to insulin. These ⁇ abnormalities lead to alterations in the metabolism of charbohydrates, lipids, ketones, and amino acids; the central feature of the syndrome is hyperglycemia.
• corticosteroids (carbohydrate metabolism- regulating) and mineralocorticoid (electrolyte balance-regulating).
• hydrocortisone (cortisol) is the main glucocorticoid
• aldosterone is the main mineralocorticoid.
• corticosteroids are numerous and diverse and include: alterations in carbohydrate, protein, and lipid metabolism; maintenance of fluid and electrolyte balance; and preservation of normal function of the cardiovascular system, the immune system, the kidney, skeletal muscle, the endocrine system, and the nervous system.
• CSH corticotropin-releasing hormone
• ACTH adrenocorticotropic hormone
• ACTH stimulates the adrenal cortex to secrete glucocorticoids, mineralocorticoids and weak androgens such as . androstenedione and dehydroepiandrosterone, which can then be converted peripherally into more potent androgens. . . .
• the testes have two main functions: synthesizing testosterone, the primary male sex hormone (androgen), and producing sperm.
• the testes may be underactive — a condition called male hypogonadism — because the pituitary gland does not secrete the hormones that are needed to stimulate the testes or because there is a problem within the testes.
• androgen production is deficient. Growth and sexual development may be retarded, sperm production is low, and the penis may be small. The symptoms vary depending on the age at which the androgen deficiency begins.
• Estrogens and progestins are endogenous hormones that produce numerous physiological actions. In women, these include, developmental effects, neuroendocrine actions involved in the control of ovulation, the cyclical preparation of the reproductive tract for fertilization and implantation, and major actions on mineral, charbohydrate, protein, and lipid metabolism.
• the ovaries are the principal source of circulating estrogen in premenopausal women. In men and postmenopausal women, the principal source of estrogen is adipose tissue stroma where estrone is synthesized from dehydroepiandrosterone, secreted by the adrenal cortex. Progesterone is secreted by the ovary mainly from the corpus luteum during the second half of the menstrual cycle. Progesterone is important for the maintenance of pregnancy.
• Recombinant somatropin (HUMATROPETM) is identical to the hormone isolated from human pitutaries.
• Corticosteroids e.g., prednisone, fludrocortisone or cortisol
• Addison's disease are useful in treatment of Addison's disease.
• hypothyroidism is treated by replacing the deficient thyroid hormone, using one of several different oral preparations.
• the preferred form is synthetic thyroid hormone, T4.
• Gonadotropins are increasingly being used for treatment of infertility. In males with hypogonadotropic hypogonadism, gonadotropin therapy can be used to achieve fertility.
• CG therapy can be used for cases of cryptorchidism (the failure of one or both testes to descend into the scrotum) that are not a result of anatomical blockage.
• Antagonists of CG are being explored for use in long term contraception methods in females. Insulin is the mainstay for treatmen of virtually all type I diabetes mellitus patients and many type II diabetes mellitus patients. Accordingly, there exists a need to identify and utilize novel proteins associated with the endocrine system and/or hormones and/or hormone antagonists.
• pancreas e.g., diabetes mellitus, diabetes insipidus, and congenital pancreatic agenesis
• adrenal glands e.g., Addison's Disease, Cushing's Syndrome, and pheochromocytomas
• ovaries e.g., ovarian cancer, polycystic ovary syndrome
• the pituitary gland e.g., hyper-, hypopituitarism, pituitary dwarfism, and pituitary adenoma
• the thyroid e.g., hyper-, hypothyroidism, Plummer's Disease, and Grave's Disease
• parathyroid gland e.g., hyper-, hypoparathyroidism
• hypothalamus and testes (e.g., Klinefelter's Syndrome, and testicular cancer).
• the present invention relates tq novel proteins. More specifically, isolated nucleic acid molecules are provided encoding novel polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing, treating, preventing and/or prognosing disorders .related to these novel polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further relates to methods and/or compositions for inhibiting or enhancing the production and function ofthe polypeptides ofthe present invention.
• the first column provides the gene number in the application for each clone identifier.
• the second column provides a unique clone identifier, "Clone ID NO:Z", for a cDNA clone related to each contig sequence disclosed in Table IA.
• the third column provides a unique contig identifier, "Contig ID:” for each of the contig sequences disclosed in Table 1 A.
• the fourth column provides the sequence identifier, "SEQ ID NO:X", for each o the contig sequences disclosed in Table IA.
• the fifth column provides the location (i.e. nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:X that delineate the preferred open reading frame (ORF) that encodes the amino acid sequence shown in the sequence listing and referenced in Table IA as SEQ ID NO:Y (column 6).
• Column 7 lists residues comprising predicted epitopes contained in the polypeptides encoded by each of the preferred ORFs (SEQ ID-NO:Y).
• polypeptides of "the invention comprise, or alternatively consist of, one, two, three, four, five or more of the predicted epitopes described in Table IA.
• Tissue Distribution shows the expression profile of tissue, cells, and/or cell line libraries which express the polynucleotides of the invention.
• the first number in column 8 represents the tissue/cell source identifier code corresponding to the key provided in Table 4. Expression of these polynucleotides was not observed in the other tissues and/or cell libraries tested.
• the second number in column 8 represents the number of times a sequence corresponding to the reference polynucleotide sequence (e.g., SEQ ID NO:X) was identified in the tissue/cell source.
• tissue/cell source identifier codes in which the first two letters are "AR” designate information generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines.
• Probe synthesis was performed in the presence of 33 P dCTP, using oligo(dT) to prime reverse transcription. After hybridization, high stringency washing conditions were employed to remove nonspecific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A local background signal subtraction was performed before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after "[array code]:" represents the mean ofthe duplicate values, following background subtraction and probe normalization.
• PSL Phosphor Stimulating Luminescence
• OMIMTM derived from Online Mendelian Inheritance, in Man (Online Mendelian Inheritance in Man, OMIMTM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD) 2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/). If the putative chromosomal location of the Query overlaps with the chromosomal location of a Morbid Map entry, an OMIM identification number is disclosed in column 10 labeled "OMIM Disease Reference(s)". A key to the OMIM reference identification numbers is provided in Table 5.
• the first column provides a unique clone identifier, "Clone ID NO:Z”, for a cDNA clone related to each contig sequence.
• the second column provides the sequence identifier, "SEQ ID NO:X”, for each contig sequence.
• the third column provides a unique contig identifier, "Contig ID:” for each contig sequence.
• the fourth column provides a BAC identifier "BAC ID NO: A” for the BAC clone referenced in the corresponding row of the table.
• the fifth column provides the nucleotide sequence identifier, "SEQ ID NO:B" for a fragment of the BAC clone identified in column four of the corresponding row ofthe table.
• the sixth column provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).
• Table 2 summarizes homology and features of some of the polypeptides of the invention.
• The. first column provides a unique clone identifier, "Clone ID NO:Z”, corresponding to a cDNA clone disclosed in Table IA.
• the second column provides the unique contig identifier, "Contig ID:” corresponding to contigs in Table 1 A and allowing for correlation with the information in Table IA.
• the third column provides the sequence identifier, "SEQ ID NO:X”, for the contig polynucleotide sequence.
• the fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined.
• NR non-redundant protein database
• PFAM protein families
• polypeptides ofthe invention comprise, or alternatively consist of, an amino acid sequence encoded by a polynucleotide in SEQ ID NO:X as delineated in columns 8 and 9, or fragments or variants thereof.
• Table 3 provides polynucleotide sequences that may be disclaimed according to certain embodiments of the invention.
• the first column provides a unique clone identifier, "Clone ID”, for a cDNA clone related to contig sequences disclosed in Table IA.
• the second column provides the sequence identifier, "SEQ ID NO:X”, for contig sequences disclosed in Table IA.
• the third column provides the unique contig identifier, "Contig ID:”, for contigs disclosed in Table 1 A.
• the fourth column provides a unique integer 'a' where 'a' is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X
• the fifth column provides a unique integer 'b' where 'b' is any integer between 15 and the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ. ID NO:X, and where b is greater than or equal to a + 14.
• the uniquely defined integers can be substituted into the general formula of a-b, and used to describe polynucleotides which may be preferably excluded from the invention.
• preferably excluded from the invention are at least one, two, three, four, five, ten, or more of the polynucleotide sequence(s) having the accession number(s) disclosed in the sixth column of this Table (including for example, published sequence in connection with a particular BAC clone).
• preferably excluded from the invention are the specific polynucleotide sequence(s) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC clone).
• Table 4 provides a key to the tissue/cell source identifier code disclosed in Table
• tissue/cell source identifier code disclosed in Table IA
• Column _8. .Columns 2-5 provide a description of the tissue or cell source. Codes corresponding to diseased tissues are indicated in' column 6 with the word "disease". The use of the word "disease" in column 6 is non-limiting.
• the tissue or cell source may be specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a normal portion of a diseased organ).
• tissues and/or cells lacking the "disease" designation may still be derived from sources directly or indirectly involved in a disease state or disorder, and therefore may have a further utility in that disease state or disorder.
• column 7 identifies the vector used to generate the library. [31]
• Table 5 provides a key to the OMIM reference identification numbers disclosed in
• Table IA column 10.
• OMIM reference identification numbers (Column 1) were derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine, (Bethesda, MD) 2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/).
• Column 2 provides diseases associated with the cytologic band disclosed in Table IA, column 9, as determined using the Morbid Map database.
• Table 6 summarizes ATCC Deposits, Deposit dates, and ATCC designation numbers of deposits made with the ATCC in connection with the present application.
• Table 7 shows the cDNA libraries sequenced, and ATCC designation numbers and vector information relating to these cDNA libraries.
• Table 8 provides a physical characterization of clones encompassed by the invention.
• the first column provides the unique clone identifier, "Clone ID NO:Z", for certain cDNA clones ofthe invention, as described in Table IA.
• the second column provides the size ofthe cDNA insert contained in the corresponding cDNA clone.
• isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
• an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or. particular cell is not the original environment of the polynucleotide.
• isolated does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features ofthe polynucleotide/sequences of the present invention.
• a "polynucleotide” refers to a molecule haying a nucleic acid sequence encoding SEQ ID NO:Y or a fragment or variant thereof; a nucleic acid sequence contained in SEQ ID-NO:X (as described in column 3 of Table IA) or the complement thereof; a cDNA sequence contained in Clone ID NO:Z (as described in column 2 of Table 1 A and contained within a library deposited with the ATCC); a nucleotide sequence encoding the polypeptide encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table IB or a fragment or variant thereof; or a nucleotide coding sequence in SEQ ID NO:B as defined in column 6 of Table IB or the complement thereof.
• the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5' and 3 1 untranslated sequences, the coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
• a "polypeptide” refers to a molecule having an amino acid sequence encoded by a polynucleotide of the invention as broadly defined (obviously excluding poly-Phenylalanine or poly-Lysine peptide sequences which result from translation of a polyA tail of a sequence corresponding to a cDNA).
• SEQ ID NO:X was often generated by overlapping sequences contained in multiple clones (contig analysis).
• a representative clone containing all or most of the sequence for SEQ ID NO:X is deposited at Human Genome Sciences, Inc. (HGS) in a catalogued and archived library.
• HGS Human Genome Sciences, Inc.
• each clone is identified by a cDNA Clone ID (identifier generally referred to herein as Clone ID NO:Z).
• Clone ID NO:Z identifier generally referred to herein as Clone ID NO:Z.
• Each Clone ID is unique to an individual clone and the Clone ID is all the information needed to retrieve a given clone from the HGS library.
• ATCC American Type Culture Collection
• Library names contain four characters, for example, "HTWE.”
• the name of a cDNA clone (Clone ID) isolated from that library begins with the same four characters, for example "HTWEP07".
• Table 1 A correlates the Clone ID names with SEQ ID NO:X.
• SEQ ID NO:X the Clone ID names with SEQ ID NO:X.
• Tables 1, 6 and 7 the Clone ID names with SEQ ID NO:X.
• the ATCC is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA.
• the ATCC deposits were made p ⁇ rsuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
• the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but.are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length.
• polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron.
• the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
• a "polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, or the complement thereof (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments described herein), the polynucleotide sequence delineated in columns 8 and 9 of Table 2 or the complement thereof, and/or cDNA sequences contained in Clone ID NO:Z (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments, or the cDNA clone within the pool of cDNA clones deposited with the ATCC, described herein), and/or the polynucleotide sequence delineated in column 6 of Table IB or the complement thereof.
• “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C in a solution comprising 50% formamide, -5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65 degree C.
• nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature.
• washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC).
• salt concentrations e.g. 5X SSC.
• variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments.
• Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
• the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
• polynucleotide which hybridizes only to pblyA ⁇ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any dbuble-stranded cD A.clone generated using oligo dT as a primer).
• the polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
• polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
• polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
• a polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
• Modified bases include, for example, tritylated bases and unusual bases such as inosine.
• a variety of modifications can be made to DNA and RNA; thus, "polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
• the polypeptide ofthe present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
• the polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
• polypeptides may be branched, for example, as a result of ubiquitination, and. they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
• Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to ' proteins such as arginylation, and ubiquitination.
• SEQ ID NO:X refers to a polynucleotide sequence described, for example, in
• SEQ ID NO:Y refers to a polypeptide sequence described in column 6 of Table IA.
• SEQ ID NO:X is identified by an integer specified in column 4 of Table IA.
• the polypeptide sequence SEQ ID NO:Y is a translated open reading frame (ORF) encoded by polynucleotide SEQ ID NO:X.
• Clone ID NO:Z refers to a cDNA clone described in- column 2 of Table IA.
• a polypeptide having functional activity refers to a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide for binding) to an anti- polypeptide antibody], immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.
• the polypeptides of the invention can be assayed for functional activity (e.g.
• a polypeptide having biological activity refers to a polypeptide exhibiting activity similar to, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).
• the first column in Table IA provides the gene number in the application corresponding to the clone identifier.
• the second column in Table IA provides a unique "Clone ID NO:Z" for a cDNA clone related to each contig sequence disclosed in Table IA.
• This clone ID references the cDNA clone which contains at least the 5' most sequence of the assembled contig and at least a portion of SEQ ID NO:X was determined by directly sequencing the referenced clone.
• the reference clone may have more sequence than described in the sequence listing or the clone may have less. In the vast majority of cases, however, the clone is believed to encode a fulMength polypeptide. In the case where a clone is not full-length, a full-length cDNA can be obtained by methods described elsewhere herein.
• the third column in Table IA provides a unique "Contig ID” identification for each contig sequence.
• the fourth column provides the "SEQ ID NO:” identifier for each of the contig polynucleotide sequences disclosed in Table IA.
• the fifth column, "ORF (From- To)" provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence "SEQ ID NO:X” that delineate the preferred open reading frame (ORF) shown in the sequence listing and referenced in Table IA, column 6, as SEQ ID NO:Y.
• the preferred ORF is the reverse complement ofthe referenced polynucleotide sequence.
• the sixth column in Table IA provides the corresponding SEQ ID NO:Y for the polypeptide sequence encoded by the preferred ORF delineated in column 5.
• the invention provides an amino acid sequence comprising, or alternatively consisting of, a polypeptide encoded by the portion of SEQ ID NO:X delineated by "ORF (From-To)". Also provided are polynucleotides encoding such amino acid sequences and the complementary strand thereto.
• polypeptides ofthe invention comprise, or alternatively consist of, at least one, two, three, four, five or more of the predicted epitopes as described in Table IA.
• Column 8 in Table 1 A provides an expression profile and library code: count for each of the contig sequences (SEQ ID NO:X) disclosed in Table IA, which can routinely be combined with the information provided in Table 4 and used to determine the tissues, cells, and/or cell line libraries which predominantly express the polynucleotides of the invention.
• the first number in column 8 represents the tissue/cell source identifier code corresponding to the code and description provided in Table 4.
• the second number in column 8 represents the number of times a sequence corresponding to the reference polynucleotide sequence was identified in the tissue/cell source.
• tissue/cell source identifier codes in which the first two letters are "AR” designate information generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines. Probe synthesis was performed in the presence of 33 P dCTP, using oligo(dT) to prime reverse transcription.
• the first column provides a unique clone identifier, "Clone ID NO:Z", for a .cDNA clone related to each contig sequence.
• the second column provides the sequence . identifier, "SEQ ID NO:X”, for each contig sequence.
• the third column provides a unique contig identifier, "Contig ID:” for each contig sequence.
• the fourth column provides a BAC identifier "BAC ID NO: A” for the BAC clone referenced in the corresponding row of the table.
• the fifth column provides the nucleotide sequence identifier, "SEQ ID NO:B” for a fragment ofthe BAC clone identified in column four of the corresponding row of the table.
• the sixth column provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).
• Table 2 further characterizes certain encoded polypeptides of the invention, by providing the results of comparisons to protein and protein family databases.
• the first column provides a unique clone identifier, "Clone ID NO:”, corresponding to a cDNA clone disclosed in Table IA.
• the second column provides the unique contig identifier, "Contig ID:” which allows correlation with the information in Table 1 A.
• the third column provides the sequence identifier, "SEQ ID NO:”, for the contig polynucleotide sequences.
• the fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined.
• the fifth column provides a description of the PFAM/NR hit identified by each analysis.
• NR ⁇ _protein database
• PFAM protein families
• the NR database which comprises the NBRF PIR database, the NCBl GenPept database, and the SIB SwissProt and TrEMBL databases, was made non-redundant using the computer program nrdb2 (Warren Gish, Washington University in Saint Louis).
• nrdb2 Warren Gish, Washington University in Saint Louis.
• Each of the polynucleotides shown in Table IA, column 3 e.g., SEQ ID NO:X or the 'Query' sequence
• the computer program BLASTX was used to compare a 6-frame translation of the Query sequence to the NR database (for information about the BLASTX algorithm please see Altshul et al., J. Mol. Biol. 215:403- 410 (1990); and Gish and States, Nat.
• the percent identity is determined by dividing the number of exact matches between the two aligned sequences in the HSP, dividing by the number of Query amino acids in the HSP and multiplying by 100.
• the polynucleotides of SEQ ID NO:X which encode the polypeptide sequence that generates an HSP are delineated by columns 8 and 9 of Table 2. ' [62] The PFAM database, PFAM version 2.1, (Sonnhammer et al, Nucl. Acids Res.,
• HMM Hidden Markov Model
• a HMM derived from PFAM version 2.1 was said to be a significant match to a polypeptide of the invention if the score returned by HMMER 1.8 was greater than 0.8 times the HMMER 1.8 score obtained with the most distantly related known member of that protein family.
• the description ofthe PFAM family which shares a significant match with a polypeptide of the invention is listed in column 5 of Table 2, and the database accession number of the PFAM hit is provided in column 6.
• Column 7 provides the score returned by HMMER version 1.8 for the alignment.
• Columns 8 and 9 delineate the polynucleotides of SEQ ID NO:X which encode the polypeptide sequence which show a significant match to a PFAM protein family. [63] .
• the invention provides a protein comprising, or alternatively consisting of, a polypeptide encoded by the polynucleotides of SEQ ID NO:X delineated in columns 8 and 9 of Table 2. Also provided are polynucleotides encoding such proteins, and the complementary strand thereto. - . '
• nucleotide sequence SEQ ID NO:X and the translated SEQ ID NO:Y are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below.
• the nucleotide sequences of SEQ ID NO:X are useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in Clone ID NO:Z. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling immediate applications in chromosome mapping, linkage analysis, tissue identification and/or typing, and a variety of forensic and diagnostic methods of the invention.
• polypeptides identified from SEQ ID NO:Y may be used to generate antibodies which bind specifically to these polypeptides, or fragments thereof, and/or to the polypeptides encoded by the cDNA clones identified in, for example, Table 1 A.
• DNA sequences generated by sequencing reactions can contain sequencing errors.
• the errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence.
• the erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence.
• the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
• the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and a predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing cDNA Clone ID NO:Z (deposited with the ATCC on October 5, 2000, and receiving. ATCC designation numbers PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and having depositor reference numbers TS-1, TS-2, AC-1, and AC-2; and/or as set forth, for example, in Table IA, 6 and 7).
• the nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited. clone in accordance with known methods. Further, techniques known in the art can be used to . verify the nucleotide sequences of SEQ ID NO:X.
• amino acid, sequence of the protem encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
• Partial cDNA clones can be made full-length by utilizing the rapid amplification of cDNA ends (RACE) procedure described in Frohman, M.A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988).
• RACE rapid amplification of cDNA ends
• RNA Poly A+ or total RNA is reverse transcribed with Superscript II (Gibco/BRL) and an antisense or complementary primer specific to the cDNA sequence.
• the primer is removed from the reaction with a Microcon Concentrator (Amicon).
• the first-strand cDNA is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL).
• the second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing three adjacent restriction sites (Xhol, Sail and Clal) at the 5' end and a primer containing just these restriction sites.
• This double-stranded cD A is PCR amplified for 40 cycles with the same primers as well as a nested cDNA-specific antisense primer.
• the PCR products are size-separated on an ethidium bfomide-agarose gel and the region of gel containing cDNA products the predicted size of missing protein-coding DNA is removed.
• cDNA is purified from the agarose with the Magic PCR Prep kit (Promega), restriction digested with Xhol or Sail, and ligated to a plasmid such as pBluescript SKII (Stratagene) at Xhol and EcoRV sites.
• This DNA is transformed into bacteria and the plasmid clones sequenced to identify' the correct protein-coding inserts. Correct 5' ends are confirmed by comparing this sequence with the putatively identified homologue and overlap with the partial cDNA clone. Similar methods known in the art and or commercial kits are used to amplify and recover 3' ends.
• kits are commercially available for purchase. Similar reagents and methods to those above are supplied in kit form from Gibco/BRL for both 5' and 3' RACE for recovery of full length genes. A second kit is available from Clontech which is a modification of a related technique, SLIC (single-stranded ligation to single- stranded cDNA), developed by Dumas et al, Nucleic Acids Res., 19:5227-32 (1991). The major differences in procedure are that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligase is used to join a restriction site-containing anchor primer to the first-strand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to sequence past.
• SLIC single-stranded ligation to single- stranded cDNA
• An alternative- to generating 5' or 3' cD A from RNA is to use cDNA library double-stranded DNA.
• An asymmetric PCR-amplified antisense cDNA strand is synthesized with an antisense cDNA-specific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA- specific antisense primer and the plasmid-anchored primer.
• RNA oligonucleotide is ligated to the 5' ends of a population of RNA presumably containing full-length gene RNA transcript and a primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest, is used to PCR amplify the 5' portion of the desired full length gene which may then be sequenced and used to generate the full length gene.
• This method starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure.
• the RNA preparation may then be treated with phosphatase if necessary to eliminate 5' phosphate groups on degraded or damaged RNA . which may interfere with the later RNA ligase step.
• the phosphatase if used is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order, to remove the cap structure present at the 5' ends of messenger RNAs.
• This reaction leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.
• This modified RNA preparation can then be used as a template for first strand cDNA synthesis using a gene specific oligonucleotide.
• the first strand synthesis - reaction can then be used as a template for PCR amplification of the desired 5' end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest.
• the resultant product is then sequenced and analyzed to confirm that the 5' end sequence belongs to the relevant gene.
• the present invention also relates to vectors or plasmids which include such
• the material deposited with the ATCC (deposited with the ATCC on October 5, 2000, and receiving ATCC designation numbers PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and receiving ATCC designation numbers TS-1, TS-2, AC-1, and AC-2; and/or as set forth, for example, in Table 1 A, Table 6, or Table 7) is a mixture of cDNA clones derived from a variety of human tissue and cloned in either a plasmid vector or a phage vector, as described, for example, in Table 7. These deposits are referred to as "the deposits" herein.
• the tissues from which some ofthe clones were derived are listed in Table 7, and the vector in which the corresponding cDNA is contained is also indicated in Table 7.
• the deposited material includes cDNA clones corresponding to SEQ ID NO:X described, for example, in Table 1 A (Clone ID NO:Z).
• a clone which is isolatable from the ATCC Deposits by use of a sequence listed as SEQ ID NO:X may include the entire coding region of a human gene or in other cases such clone may include a substantial portion of the coding region of a human gene.
• sequence listing may in some instances list only a portion of the DNA sequence in a clone included in the ATCC Deposits, it is well within the ability of one skilled in the art to sequence the DNA included in a clone contained in the ATCC Deposits by use of a sequence .(or portion thereof) described in, for example Tables lAor 2 by procedures hereinafter further described, and others apparent to those skilled in the art.
• Table 7 Also provided in Table 7 is the name of the vector which contains the cDNA clone. Each vector is routinely used in the art. The following additional information is provided for convenience.
• pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene.
• Phagemid pBS may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Express vector. Both phagemfds may be transformed into E. coli strain XL-1 Blue, also available from Stratagene.
• Vectors pSportl, pCMVSport 1.0, pCMVSport 2.0 arid pCMVSport 3.0 were obtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, MD 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from Life Technologies. . See, for instance, Gruber, C. E., et al., Focus 15:59- (1993). Vector lafmid BA (Bento Soares, Columbia University, New York, NY) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue.
• Vector pCR ® 2.1 which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, CA 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. See, for instance, Clark, J. M., Nuc. Acids Res. 76:9677-9686 (1988) and Mead, D. et al, Bio/Technology 9: (1991). [76] The present invention also relates to " the genes corresponding to SEQ ID NO:X,
• SEQ ID NO:Y SEQ ID NO:Y
• the deposited clone Clone ID NO:Z
• the corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
• allelic variants, orthologs, and/or species homologs are also provided in the present invention. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X or the complement thereof, polypeptides encoded by genes corresponding to SEQ ID NO:X or the complement thereof, and/or the cDNA contained in Clone ID NO:Z, using information from the sequences disclosed herein or the clones deposited with the ATCC.
• allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
• polypeptides of the invention can be prepared in any suitable manner.
• Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
• the polypeptides may be iri the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production. [80] .
• the polypeptides ofthe present invention are preferably provided in an isolated form; and preferably are substantially purified.
• a recombinantly produced version of a polypeptide, including the secreted polypeptide can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one- step method described in Smith and Johnson, Gene 67:31-40 (1988).
• Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the polypeptides of the present invention in methods which are well known in the art.
• the present invention provides a polynucleotide comprismg, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA sequence contained in Clone ID NO:Z.
• the present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X or a complement thereof, a polypeptide encoded by the cDNA contained in Clone ID NO:Z, and/or the polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table IB.
• Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, a polypeptide encoded by the cDNA contained in Clone ID NO:Z, and/or a polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table IB are also encompassed by the invention.
• the present invention further encompasses a polynucleotide comprising, or alternatively consisting of, the complement of the nucleic acid sequence of SEQ ID NO:X, a nucleic acid sequence, encoding a polypeptide encoded by the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA contained in Clone ID NO:Z.
• a polynucleotide comprising, or alternatively consisting of, the complement of the nucleic acid sequence of SEQ ID NO:X, a nucleic acid sequence, encoding a polypeptide encoded by the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA contained in Clone ID NO:Z.
• representative examples of polynucleotides ofthe inventiori comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in Table IB column 6, or any combination thereof.
• polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the. sequences delineated in Table IB column 6, or any combination thereof.
• the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table IB, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table IB, column 5).
• the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table IB, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table IB, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table IB, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table IB, column 4).
• Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
• representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more ofthe sequences delineated in column 6 of Table IB which correspond to the same Clone ID NO:Z (see Table IB, column 1), or any combination thereof.
• Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in column 6 of Table IB which correspond to the same Clone ID NO:Z (see Table IB, column 1), or any combination thereof.
• the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table IB which correspond to the same Clone ID NO:Z (see Table IB, column 1) and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table ⁇ B, column 5).
• polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table IB which correspond to the same Clone ID NO:Z (see Table IB, column 1) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO: A (see Table IB, column 4).
• the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table IB which correspond to the same Clone ID NO:Z (see Table IB, column 1) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table IB, column 4).
• Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
• polynucleotides ofthe invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table IB which correspond to the same contig sequence identifer SEQ ID NO:X (see Table IB, column 2), or any combination thereof.
• polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in column 6 of Table IB which correspond to the same contig sequence identifer SEQ ID NO:X (see Table IB, column 2), or any combination thereof.
• the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table IB which correspond to the same contig sequence identifer SEQ ID NO:X (see Table IB, column 2) and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table IB, column 5).
• the above-described polynucleotides of the invention comprise, or .
• the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table IB which correspond to the same contig sequence identifer ' SEQ ID ⁇ NO:X (see Table IB, column 2) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (See Table IB, column 4).
• Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants ofthe above-described polynucleotides and polypeptides are also encompassed by the invention.
• representative examples of polynucleotides ofthe invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of Table IB column 6, or any combination thereof: Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in the same row of Table IB column 6, or any combination thereof.
• the polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in the same row of Table IB column 6, wherein sequentially delineated sequences in the table (i.e. corresponding to those exons located closest to each other) are directly contiguous in a 5' to 3' orientation.
• above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table IB, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table IB, column 5).
• the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table IB, column 6, and. have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table IB, column 4).
• polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table IB, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table IB, column 4).
• Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that-bind these polypeptides are also encompassed by the invention.
• polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more ofthe sequences delineated in column 6 of Table IB:, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table IB, column 2) or fragments or variants thereof.
• Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
• polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more ofthe sequences delineated in column 6 of Table IB which correspond to the same Clone ID NO:Z (see Table IB, column 1), and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1 A or IB) or fragments or variants thereof.
• the delineated sequence(s) and polynucleotide sequence of SEQ ID NO:X correspond to the same Clone ID NO:Z.
• Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
• polynucleotides of the invention comprise, or alternatively -consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of column 6 of Table IB, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table IA or IB) or fragments or variants thereof.
• the delineated sequence(s) and polynucleotide sequence of SEQ ID NO:X correspond to the same row of column 6 of Table IB.
• polynucleotides ofthe invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table IB and the 5' 10 polynucleotides of the sequence of SEQ ID NO:X are directly contiguous.
• Nucleic acids which hybridize to the complement of these .20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention.
• Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• polynucleotides ofthe invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table IB and the 5' 10 polynucleotides of a fragment or variant ofthe sequence of SEQ ID NO:X are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention.
• Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides ofthe sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table IB are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also - encompassed by. the invention.
• Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table IB are directly contiguous.
• Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention.
• Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants ofthe above-described polynucleotides, nucleic acids, and polypeptides, .are also encompassed by the invention. .
• polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table IB and the 5' 10 polynucleotides of another sequence in column 6 are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention.
• Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table IB and the 5' 10 polynucleotides of another sequence in column 6 corresponding to the same Clone ID NO:Z (see Table IB, column 1) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 lower stringency conditions, are also encompassed by the invention.
• polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one sequence in column 6 corresponding to the same contig sequence identifer SEQ ID NO:X (see Table IB,.
• column 2 are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded r by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments ' and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table IB and the 5' 10 polynucleotides of another sequence in column 6 . corresponding -to the same row are directly contiguous.
• the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table IB is directly contiguous with the 5' 10 polynucleotides of the next sequential exon delineated. in Table IB, column 6.
• Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention.
• Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention, Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• each contig sequence (SEQ ID NO:X) listed in the fourth column of Table IA preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, b is an integer of 15 to the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a + 14.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a and b are integers as defined in columns 4 and 5, respectively, of Table 3.
• the polynucleotides of the invention do not consist of at least one, two, three, four, five, ten, or more of the specific polynucleotide sequences referenced by the Genbank Accession No. as disclosed in column 6 of Table 3 (including for example, published sequence in connection with a particular BAC clone).
• preferably excluded from the invention are the specific polynucleotide sequence(s) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in ah identified BAC clone). In no way is this listing meant to encompass all ofthe sequence ' s which may be excluded by the general formula, it is just a representative example. All references available through these accessions are hereby incorporated by reference in their entirety. TABLE 3
• AR066488 A82595, AR016514, AR060138, A45456, A26615, AR052274, Y09669, AR060385, A85396, AR066482, A44171, AB002449, AR066487, X68127, A43192, A43190, AR038669, A30438, A85477, D50010, 119525, A86792, X93549, Y17187; AR066490, 118367, U87250, D88507, A63261, A70867, AR008408, AR062872, D 13509, AR060133, AR016691, AR016690, U46128, A64136, A68321, 114842, AR054175, AF135125, AF123263, AR050680, U79457, X93535, U87247, and AR008382.
• HWHHW5 31 1112445 1 - 1418 15 - 1432 AW192697, AI336274, AI041819, AA808430, 4 ' AA780044, AI740785, AI073467, AA883903, AI022809, AW157214, AI871953, AI521233, AW009412, AA554447, AA443442, AA689601, AA8867.25, AI589839, AA863294, AI034240, AA595998, AI669406, AA932317, AI986294, AW152255, AI925407, AA806498, AI887353, AA978039, AI338458, AI042442i AA443308, AI537869, AA890338, AA992867, AI139401, AI085301, AI263340, AA917926, N58734, AI348041, AA854105, AI742678, AI080542
• HAPQS74 55 1161259 1 - 5157 15 - 5171 AW070235, AA481601, AW237310, AI004312, AA287561, AI401739, AA889952, AI743497, AI905471, AI627341, AA918798, AI198899, AI807434, AW004921, AA287878, AA040134, AA291780, AA040087, AI090624, AI014512, W47451, AA991326, AA192275, AA335655, AA195630, AW002298, AA437120, R07941, AW069632, AI271814, T12458, AA811669, AA552246, AA281173, R06392, F25492, AA775242, AA931765, R06391, F19391, T59746, R31819, AA732918, T85620, T30711,
• AF090900 AF137367, AL137711, S79832, ALl 10222, AF022363, A18777, X66862, J05032, 189947, 148978, AL133558, AL049314, A08913, AF185576, AR038854, AB025103, A08912, A27171, A08910, AL137476, 189931, A08909, AL049460, ALl 17585, E02221, 149625, A08908.
• HNTMB90 66 1154792 1 - 1644 15 - 1658 AW242587, and AA496178.
• HPMLD30 78 1223486 1 - 2860 15 - 2874 AA653812, S78429, E03539, and X01966.
• H7TSA07 95 1128629 1 - 521 15 - 535 AA077171, AR028893, AR028901, AR028894, AR028902, D82935, AC006466, L16506, L16680, AR028891, Z23274, Z23282, AR028899, D14909, D16465, AR028888, Z23272, Z23275, D17290, AR028892, AR028900, D17516, AR028895, AR028903, AR028890, Z23279, AR028898, D14908, Z23273, AR028889, and AR028897.
• HSLHC11 100 966325 1 - 244 ' 15 - 258 AA585098, T18597, R45895, R28735, R29445, AA585325, AA585476, R29218, R28892, AA170832, R29657, R28967, R28965, AA283326, D60765, AA585101, D53447, Z32887, D59751, C15406, D54897, D53161, C16315, D60844, D57491, C16293, R28895, D53472, AI557734, D61254, Z32822, C15069, AA585439, Z28355, AI557262, AI546875, AI546999, C16294, C15120, D52835, AI541383, AI557864, AI541356, D59436, AA585155, D61185, C16292, AI557763, AI547006, AI547250, AI526140,
• HE9TK49 108 1126487 1 - 1441 15 - 1455.
• HCQCI06 114 1082633 1 - 1982 15 - 1996 AI827458, AI627268, AI654812, AI123952, AI093158, AI399783, AI419376, AI338834, AI308129, AI800234, AI082388, AW022777, AI751915, AA256582, AI675002, AW015280, W78050, AI962653; AA405577, AA405578, AI247176, H28492, AA636051, AI141051, AI751916, AI633550, AA318231, AA143272, AI081761, AI819602, D62975, AI379282, AA852344, AA343597, AI143558, AA625818, AI378667, W79144, AA770096, AI867823, W81546, AA938549, AI421066, AA897387, AI219502, AI925096, AI088303, D
• HWLXT48 123 1152282 1 - 734 15 - 748 AW449148, AA932449, AW245862, AA306873, N44651, AA305881, AA088641, N31513, AA906040, R25850, R88663, AI073401, AI824292, AW248398, AA137171, AW189250, AI952811, AL136295, and AF044127.
• HBCPT10 124 1152325 1 - 941 15 - 955 - AW449148, AA932449, AA306873, AW245862, N44651, AA305881, AA088641, N31513, AA906040, R25850, R88663, AI073401, AI824292, AW248398, AA137171, AL136295, and AF044127.
• AI826276 AH 89999, H59895, R76230, R31547, AI001978, H72421, N39659, R02720, R73757, R73883, R73670, AA483809, R63227, AA885048, AW104374, AA295914, H02478, AI885432, AI784120, AI149979, AA442745, R73969, R37557, H03862, R26664, H17430, AA853105, AA832163, R25112, R76229, H00834, AA833569, H03308, R79832, R36714, R31066, D60944, AI417747, AA579968, AA305581, AA429715, AA705903, R76442, AI559375, AF151810, and AF039696.
• HCBME31 130 971321 1 - 1470 15 - 1484 AW385618, AW385612, AW385608, AI394057, AA031354, AW293177, AA031353, AA369669, AH 74266, AW403498, AW376414, AAl 57797, AW376997, AW376921, AFl 14491, and AC004262.
• HJMBP48 131 1135867 1 - 1423 15 - 1437 U66688, AI420480, AI656922, AI168486, AAl 16008, AAl 16009, AW015363, AI083583, AI680780, R89046, AI825488, N22021, H67248, AA357634, AA719140, AA309016, AI989486, AI380719, AA524463, H49149, AA190206, AA585380, AI554218, AI679211, AI251830, AI306613, AI640379, AI702073, AI612750, AI867042, AI866002, AI680457, AI566630, AI927092, AA830821, AW079159, AI805638, AI249877, AW193274, AW002174, AI624668, AI919345, AI890784, AI285431, AI860537, AI919107,
• HDPBI30 133 974711 1 - 2911 15 - 2925 AA714520, N78665, W15172, AL134531, AA074818, AI251157, AI311635, AA079403, AW130754, AI935943, AF083955, AC005015, AL034423, AP000030, AC002992, AC004216, AC003013, U91321, AC003684, AC002528, ALl 17258, AL021155, AP000045, AF053356, AL033521, AC004598, U91326, AL035072, AD000091, U82668, AC012384, L44140, AF006752, AL034350, AC006039, AC005756, AC005072, AL034429, AC002352, AC005682, AC003663, AC005049, AC007298, AC005620, AC004887, ALl 17694, AC005911, AC
• the present invention is directed to variants of the polynucleotide sequence disclosed in SEQ ID NO:X or the complementary strand thereto, nucleotide sequences encoding the polypeptide of SEQ ID NO:Y, the nucleotide sequence of SEQ ID NO:X encoding the polypeptide sequence as defined in column 7 of Table IA, nucleotide sequences encoding the polypeptide as defined in column 7 of Table IA, the nucleotide sequence as defined in columns 8 and 9 of Table 2, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as ' defined in columns 8 and 9 of Table 1, the nucleotide sequence as defined in column 6 of Table IB, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in column 6 of Table IB, the cDNA sequence contained in Clone ID NO:Z, and/or nucleotide sequences encoding the polypeptide encoded by the
• the present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID NO:Y, the polypeptide sequence as defined in column 7 of Table IA, a polypeptide sequence encoded by the polynucleotide sequence in SEQ ID NO:X, a polypeptide sequence encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2, a polypeptide sequence encoded by the nucleotide sequence as defined in column 6 of Table IB, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, and/or a polypeptide sequence encoded by the cDNA sequence contained in Clone ID NO:Z.
• Variant refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide ofthe present invention.
• one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence described in SEQ ID NO:X or contained in the cDNA sequence of Clone ID NO:Z; (b) a nucleotide sequence in SEQ ID NO:X or- the cDNA in Clone ID NO:Z which encodes the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; (c) a nucleotide sequence in SEQ ID NO:X or the cDNA in Clone ID NO:Z which encodes a mature polypeptide; (d) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of Clone ID NO:Z, which encodes a biologically active fragment of a polypeptide; (e) a nucleotide
• the present invention is also directed to nucleic acid molecules which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%o, 99% or 100%, identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), or (j) above, the nucleotide coding sequence in SEQ ID NO:X or the complementary strand thereto, the nucleotide coding sequence ofthe cDNA contained in Clone ID NO:Z or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID NO:Y, a nucleotide sequence encoding a polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:X, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, a
• Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides and nucleic acids.
• the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent hybridization conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i), above, as are polypeptides encoded by these polynucleotides.
• polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions, or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
• the invention provides a purified protein comprising, or alternatively consisting of, a polypeptide having an amino acid sequence selected from the group consisting of: (a) the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; (b) the amino acid sequence of a mature form of a polypeptide having the amino acid sequence of SEQ ID NO:Y or the amino acid sequence encoded by the cDNA in Clone ID NO:Z; (c) the amino acid sequence of a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; and (d) the amino acid sequence of an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO. ⁇ or the complete amino acid sequence encoded by the cDNA.in Clone ID NO:Z.
• the present invention is also directed to proteins which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%o or 100%), identical to, for example, any of the amino acid sequences in (a), (b), (c), or (d), above, the amino acid sequence shown in SEQ ID NO:Y, the amino acid sequence encoded by the cDNA contained in Clone ID NO:Z, the amino acid sequence of the- polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table IB, the amino acid sequence as defined in column 7 of Table IA, an amino acid sequence encoded by the nucleotide sequence in SEQ ID NO:X, and an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO;X.
• polypeptides are also provided (e.g., those fragments described herein).
• Further proteins encoded by polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these amino acid sequences under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are the polynucleotides encoding these proteins.
• nucleic acid having a nucleotide sequence at least, for example, 95%> “identical" to a reference nucleotide sequence ofthe present invention it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide.
• nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
• the query sequence may be an entire sequence referred to in Table 1 A or 2 as the ORF (open reading frame), or any fragment specified as described herein.
• nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs.
• a preferred method for determining the best overall match between a query sequence (a sequence ofthe present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are both DNA sequences.
• RNA sequence can be compared by converting U's- to T's.
• the result of said global sequence alignment is expressed as percent identity.
• This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
• This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases ofthe subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
• a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity.
• the deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end.
• the 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%.
• a 90 base subject sequence is compared with a 100 base query sequence.
• deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query.
• percent identity calculated by FASTDB is not manually corrected.
• bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
• a polypeptide having an amino acid sequence at least, for example, 95%> "identical" to a query amino acid sequence of the present invention it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
• the amino acid sequence of the subject polypeptide may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
• up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid.
• These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
• any particular polypeptide is at least 80%, 85%, 90%
• Table IA e.g., the amino acid sequence identified in column 6
• Table 2 e.g., the amino acid sequence of the
• a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.6:237-245 (1990)).
• a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity.
• the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C- terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention.
• a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity.
• the deletion occurs at the N- terminus of the subject sequence and therefore, the FASTDB alignment does, not show a matching/ alignment of the first 10 residues at the N-terminus.
• the 10 unpaired residues represent 10%> of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%).
• a 90 residue subject sequence is compared with a 100 residue query sequence.
• the polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both.
• polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide are preferred.
• Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred.
• polypeptide variants in which less than 50, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred.
• Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E.
• Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism.. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-rnaturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
• variants may be generated to improve or alter the characteristics of the polypeptides of the present invention.
• one or more amino acids can be deleted from the N- terminus or C-terminus of the polypeptide of the present invention without substantial loss of biological function.
• Ron et al. J. Biol. Chem. 268: 2984-2988 (1993)
• variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues.
• Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein.
• the invention further includes polypeptide variants which show- a functional activity (e.g., biological activity) of the polypeptides of the invention.
• a functional activity e.g., biological activity
• variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
• nucleic acid molecules at least 80%, 85%, 90%), 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, (e.g., encoding a polypeptide having the amino acid sequence of an N and/or C terminal deletion), irrespective of whether they encode a polypeptide having functional activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
• PCR polymerase chain reaction
• nucleic acid molecules of the present invention that do not encode a polypeptide having functional activity include, inter alia, (1) isolating a gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern Blot analysis for detecting mRNA expression in specific tissues (e.g., normal or diseased tissues); and (4) in situ hybridization (e.g., histochemistry) for detecting mRNA expression in specific tissues (e.g., normal or diseased tissues).
• in situ hybridization e.g., histochemistry
• nucleic acid molecules having sequences at least 80%», 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, which do, in fact, encode a polypeptide having functional activity.
• a polypeptide having "functional activity” is meant, a polypeptide capable, of displaying one or more known functional activities associated with a full-length (complete) protein of the invention.
• Such functional activities mclude, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an anti-polypeptide of the invention antibody], immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or .ligand for a polypeptide ofthe invention.
• immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, ' protein A assays, and immuno electrophoresis assays, etc.
• antibody binding is detected by detecting a label on the primary antibody.
• the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
• the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope ofthe present invention.
• binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995).
• the ability of physiological correlates of a polypeptide of the present invention to bind to a substrate(s) of the polypeptide of the invention ca be routinely assayed using techniques known in the art.
• nucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, for example, the nucleic acid sequence of the cDNA contained in Clone ID NO:Z, the nucleic acid sequence referred to in Table IA (SEQ ID NO:X), the nucleic acid sequence disclosed in Table 2 (e.g,.
• nucleic acid sequence delineated in columns 8 and 9) or fragments thereof will encode polypeptides "having functional activity.”
• degenerate variants of any of these nucleotide sequences all encode the same polypeptide, in many instances, this will be clear to the skilled artisan even without performing the above described comparison assay.
• a reasonable number will also encode a polypeptide having functional activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e:g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.
• the first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protem function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity ofthe protein.
• the second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. See Cunningham and Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity.
• tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
• variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitutions with one or more of the amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability- and/or solubility of the polypeptide (for example, polyethylene glycol), (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, serum albumin (preferably human serum albumin) or a fragment thereof, or leader or secretory sequence, or a sequence facilitating purification, or (v) fusion of the polypeptide with another compound, such as albumin (including but not limited to recombinant albumin (see, e.g., U.S.
• polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al, Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).
• a further embodiment of the invention relates to polypeptides which comprise the amino acid sequence of a polypeptide having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions from a polypeptide sequence disclosed herein.
• a polypeptide prefferably has an amino acid sequence which comprises the amino acid sequence of a polypeptide of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID NO:X, an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columnns 8 and 9 of Table 2, an amino acid sequence encoded by the complement of SEQ ID NO:X, and/or an amino acid sequence encoded by cDNA contained in Clone ID NO:Z which contains, in order of ever-increasing preference, at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.
• the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of a reference amino acid sequence selected from: (a) the amino acid sequence of SEQ ID NO:Y or fragments thereof (e.g., the mature form and/or other fragments described herein); (b) the amino acid sequence encoded by SEQ ID NO:X or fragments thereof; (c) the amino acid sequence encoded by the complement of SEQ ID NO:X or fragments thereof; (d) the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or fragments thereof; and (e) the amino acid sequence encoded by cDNA contained in Clone ID NO:Z or fragments thereof; wherein the fragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, amino acid residue additions, substitutions, and/or deletions when compared to the reference amino acid sequence.
• the amino acid substitutions are conservative.
• polynucleotide fragment refers to a polynucleotide having a nucleic acid sequence which, for example: is a portion of the cDNA contained in Clone ID NO:Z or the complementary strand thereto; is a portion of the polynucleotide sequence encoding the polypeptide encoded by the cDNA contained in Clone ID NO:Z or the complementary strand thereto; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion ofthe polynucleotide sequence of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide
• the polynucleotide fragments ofthe invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length.
• a fragment "at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in Clone ID NO:Z, or the nucleotide sequence shown in SEQ ID NO:X or the complementary stand thereto. In this context "about” includes the particularly recited value or a value larger or smaller by several (5, 4, 3, 2, or 1).
• nucleotides at either terminus or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., at least 160, 170, 180, 190, 200, 250, 500, 600, 1000, or 2000 nucleotides in length ) are also encompassed by the invention.
• polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number 1-50, 51- 100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351- 1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101- 2150, 2151-2200, 2201-225.0, 225
• polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number 1-50, 51- 100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351- 1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101- 2150, 2151-2200, 2201-2250, 2251-2300
• polypeptides encode a polypeptide which has a functional activity (e.g., biological activity). More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
• polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence delineated in Table IB column 6.
• polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence that is the complementary strand of-a sequence delineated in column 6 of Table IB.
• the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table IB, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table IB, column 5).
• the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table IB, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO: A (see Table IB, column 4).
• the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated Table IB, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table IB, column 4).
• polynucleotides ofthe invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table IB, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table IB, column 2) or fragments or variants thereof.
• polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
• polynucleotides ofthe invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table IB which correspond to the same Clone ID NO:Z (see Table IB, column 1), and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table IA or IB) or fragments or variants thereof.
• Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
• polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in the same row of column 6 of Table IB, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table IA or IB) or fragments or variants thereof.
• Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
• polynucleotides ofthe invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table IB and the 5' 10 polynucleotides of the sequence of SEQ ID NO:X are directly contiguous. Nucleic acids which' hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention.
• Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• polynucleotides ofthe invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table IB and the 5' 10 polynucleotides of a fragment or variant ofthe sequence of SEQ ID NO:X (e.g., as described herein) are directly contiguous Nucleic acids which hybridize to the,- complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention.
• Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants ofthe above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3 ' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table IB are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention.
• Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table IB and the 5' 10 polynucleotides of another sequence in column 6 are directly contiguous.
• the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table IB is directly contiguous with the 5' 10 polynucleotides of the next sequential exon delineated in Table IB, column 6.
• Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention.
• Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants ofthe above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• a "polypeptide fragment” refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:Y, a portion of an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columnns 8 and 9 of Table 2, a portion of an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO:X, a portion of an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, and/or a portion of an amino acid sequence encoded by the cDNA contained in Clone ID NO:Z.
• Protein (polypeptide) fragments may be "free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
• Representative examples of polypeptide fragments of the invention include, for example, fragments comprising, or alternatively consistmg of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81- .
• polypeptide fragments of the invention include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301- 320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681- 700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960,
• polypeptide fragments of the invention may be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, or 150 amino acids in length.
• “about” includes the particularly recited ranges or values, or ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
• Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
• polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form.
• any number of amino acids can be deleted from the carboxy " terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
• the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X or the complement thereof, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, a polypeptide encoded by the portion of SEQ ID NO:B as defined in column 6 of Table IB, and/or a polypeptide encoded by the cDNA contained in Clone ID NO:Z).
• a polypeptide of SEQ ID NO:Y e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X or the complement thereof, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, a
• N-terminal deletions may be described by the general formula m-q, where q is a whole integer representing the total number of amino acid residues in a polypeptide of the invention (e.g., the polypeptide disclosed in SEQ ID NO:Y, or the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), and m is defined as any integer ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• the present invention further provides polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and or a polypeptide encoded by the cDNA contained in Clone ID NO:Z).
• C-terminal deletions may be described by the general formula 1-n, where n is any whole integer ranging from 6 to q-1, and where n corresponds to the position of amino acid residue in a polypeptide of the invention.
• polypeptides encoding these polypeptides are also encompassed by the invention.
• any of the above described N- or C-terminal deletions can be combined to produce a - and C-terminal deleted polypeptide.
• the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of a polypeptide encoded by SEQ ID NO:X (e.g., including, but not limited to, the preferred polypeptide disclosed as SEQ ID NO:Y and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), the cDNA contained in Clone ID NO:Z, and/or the complement thereof, where n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• the present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein.
• the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N- and C-terminal deletions.
• Polynucleotides encoding these polypeptides are also encompassed by the invention.
• Any polypeptide sequence encoded by, for example, the polynucleotide sequences set forth as SEQ ID NO:X or the complement thereof, (presented, for example, in Tables 1 A and 2), the cDNA contained in Clone ID NO:Z, or the polynucleotide sequence as defined in column 6 of Table IB, may be analyzed to determine certain preferred regions of the polypeptide.
• amino acid sequence of a polypeptide encoded by a polynucleotide sequence of SEQ ID NO:X may be analyzed using the default parameters of the DNASTAR computer algorithm (DNASTAR, Inc., 1228 S. Park St., Madison, WI 53715 USA; http://www.dnastar.com/).
• Polypeptide regions that may be routinely obtained using the DNASTAR computer algorithm include, but are not limited to, Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle hydrophilic regions and hydrophobic regions; Eisenberg alpha- and beta-amphipathic regions; Karplus-Schulz flexible regions; Emini surface-forming regions; and Jameson-Wolf regions of high antigenic index.
• highly preferred polynucleotides of the invention in this regard are those that encode polypeptides comprising regions that combine several structural features, such as several (e.g., 1, 2, 3 or 4) ofthe features set out above.
• Kyte-Doolittle hydrophilic regions and hydrophobic regions, Emini surface-forming regions, and Jameson-Wolf regions of high antigenic index can routinely be used to determine polypeptide regions that exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from data by DNASTAR analysis by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
• Preferred polypeptide fragments of the invention are fragments comprising, or alternatively, consisting of, an amino acid sequence that displays a functional activity (e.g. biological activity) of the polypeptide sequence of which the amino acid sequence is a fragment.
• a polypeptide displaying a "functional activity” is meant a polypeptide capable of one or more known functional activities associated with a full-length protein, such as, for example, biological activity, antigenicity, immunogenicity, and/or multimerization, as described herein.
• polypeptide fragments ' are biologically active fragments.
• Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide ofthe present invention. 'The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
• polypeptides of the invention comprise, or ' alternatively consist of, one, two, three, four, five or more of the antigenic fragments of the polypeptide of SEQ ID NO:Y, or portions thereof. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• the present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of: the polypeptide sequence shown in SEQ ID NO:Y; a polypeptide sequence encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide sequence encoded by the portion of SEQ ID NO:X as- defined in columns 8 and 9 of Table 2; the polypeptide sequence encoded by the portion of SEQ ID NO:B as defined in column 6 of Table IB or the complement thereto; the polypeptide sequence encoded by the cDNA contained in Clone ID NO:Z; or the polypeptide sequence encoded by a polynucleotide that hybridizes to the sequence of SEQ ID NO:X, the complement of the sequence of SEQ ID NO:X, the complement of a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, or the cDNA sequence contained in Clone ID NO:Z under stringent hybridization conditions or alternatively, under lower stringency hybridization as defined supra.
• the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:X, or a fragment thereof), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or alternatively, under lower stringency hybridization conditions defined supra.
• polypeptide sequence of the invention such as, for example, the sequence disclosed in SEQ ID NO:X, or a fragment thereof
• polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or alternatively, under lower stringency hybridization conditions defined supra.
• epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human.
• the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide.
• An "immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci.
• antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
• Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985) further described in U.S. Patent No. 4,631,211.)
• antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids.
• Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length.
• Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof.
• Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope.
• Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes.
• Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al. Science 219:660-666 (1983)).
• Non-limiting examples of epitopes of polypeptides that can be used to generate antibodies of the invention include a polypeptide comprising, or alternatively consisting of, at least one, two, three, four, five, six or more of the portion(s) of SEQ ID NO:Y specified in column 7 of Table IA. These polypeptide fragments have been determined to bear antigenic epitopes of the proteins of the invention by the analysis of the Jameson- Wolf antigenic index which is included in the DNAStar suite of computer programs.
• a polypeptide contains at least one, two, three, four, five, six or more of the portion(s) of SEQ ID NO:Y shown in column 7 of Table 1 A, but it may contain additional flanking residues ori either the amino or carboxyl termini of the recited portion.
• additional flanking sequences are preferably sequences naturally found adjacent to the portion; i.e., contiguous sequence shown in SEQ ID NO:Y.
• the flanking sequence may, however, be sequences from a heterolgous polypeptide, such as from another protein described herein or from a heterologous polypeptide not described herein.
• epitope portions of a polypeptide ofthe invention comprise one, two, three, or more ofthe portions of SEQ ID NO:Y shown in column 7 of Table IA.
• immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. See, for instance, Sutcliffe et al, supra; Wilson et al, supra; Chow et al, Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al, J. Gen. Virol. 66:2347-2354 (1985).
• immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic. epitopes.
• the polypeptides comprising one or more • immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier.
• immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g, in Western blotting).
• Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g, Sutcliffe et al, supra; Wilson et al, supra, and Bittle et al, J. Gen. Virol, 66:2347-2354 (1985).
• animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
• KLH keyhole limpet hemacyanin
• peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl- N- hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.
• Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal . and/or intradermal injection of emulsions containing about 100 ⁇ g of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.
• booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
• the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti- peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution ofthe selected antibodies according to methods well known in the art.
• the polypeptides of the present invention e.g, those comprising an immunogenic or antigenic epitope
• polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHI, CH2, CH3, or any combination thereof and portions thereof, resulting in chimeric polypeptides.
• polypeptides and/or antibodies of the present invention may be fused with albumin (including but hot limited to recombinant human serum albumin or fragments or variants thereof (see, e.g, U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No.
• polypeptides and/or antibodies of the present invention are fused with the mature form of human serum albumin (i.e, amino acids 1 - 585 of human serum albumin as shown in Figures 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference in its entirety.
• polypeptides and/or antibodies of the present invention are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S.
• Patent 5,766,883 herein incorporated by reference in its entirety.
• Polypeptides and/or antibodies of the present invention may be fused to either the N- or C-terminal end ofthe heterologous protein (e.g, immunoglobulin Fc polypeptide or human serum albumin polypeptide).
• the heterologous protein e.g, immunoglobulin Fc polypeptide or human serum albumin polypeptide.
• Polynucleotides encoding fusion proteins of the invention are also encompassed by the invention.
• Such fusion proteins as those described above may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting ofthe first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g, EP 394,827; Traunecker et al. Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g, insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g, PCT Publications WO 96/22024 and WO 99/04813).
• antigens e.g, insulin
• FcRn binding partner such as IgG or Fc fragments
• IgG fusion proteins that have a disulfide- linked dimeric structure due to the- IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g, Fountoulakis et al, J. Biochem, 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g, the hemagglutinin (HA) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al.
• HA hemagglutinin
• any polypeptide ofthe present invention can be used to generate fusion proteins.
• the polypeptide of the present invention when fused to a second protein, can be used as . an antigenic tag.
• Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide.
• secreted proteins target cellular locations based on trafficking signals
• polypeptides of the present invention which are shown to be secreted can be used as targeting molecules once fused to other proteins.
• domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions.
• the fusion does not necessarily need to be direct, but may occur through linker sequences.
• proteins of the invention are fusion proteins comprising an amino acid sequence that is an N and/or C- terminal deletion of a polypeptide of the invention.
• the invention is directed to a fusion protein comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%>, 98% or 99% identical to a polypeptide sequence ofthe invention.
• Polynucleotides encoding these proteins are also encompassed by the invention.
• fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification . . Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
• polypeptides of the present invention can be combined with heterologous polypeptide sequences.
• the polypeptides of the present invention may be fused with heterologous polypeptide sequences, for example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CHI, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), or albumin (including, but not limited to, native or recombinant human albumin or fragments or variants thereof (see, e.g, U.S. Patent No.
• EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
• the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (EP-A 0232 262).
• deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
• the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
• human proteins such as hIL-5
• Fc portions for the purpose of high- throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al, J. Molecular Recognition 8:52-58 (1995); K. Johanson et al, J. Biol. Chem. 270:9459-9471 (1995).
• the polypeptides of the present invention can be fused to marker sequences, such as a polypeptide which facilitates purification of the fused polypeptide.
• the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc, 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
• hexa-histidine provides for convenient purification of the fusion protein.
• Another peptide tag useful for purification, the "HA" tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al. Cell 37:767 (1984)).
• DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al, Curr. Opinion Biotechnol.
• alteration of polynucleotides corresponding to SEQ ID NO:X and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling.
• DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence.
• polynucleotides ofthe invention may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
• one or more components, motifs, sections, parts, domains, fragments, etc, of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
• any of these above fusions can be engineered using the polynucleotides or the polypeptides ofthe present invention.
• the present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by synthetic and recombinant techniques.
• the vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
• the polynucleotides of the invention may be joined to a vector containing a selectable marker for propagation in a host.
• a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
• the polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan.
• the expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
• the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end ofthe polypeptide to be translated.
• the expression vectors will preferably include at least one selectable marker.
• markers include dihydrofolate reductase, G418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.
• Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.
• insect cells such as Drosophila S2 and Spodoptera Sf9 cells
• animal cells such as CHO, COS, 293, and Bowes melanoma cells
• plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
• vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc.
• preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
• Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL- Sl, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlbad, CA).
• Other suitable vectors will be readily apparent to the skilled artisan.
• Glutaminase GS
• DHFR DHFR
• An advantage of glutamine synthase based vectors are the availabilty of cell lines (e.g, the murine myeloma cell line, NSO) which are glutamine synthase negative.
• Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g, Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene.
• glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657, which are hereby incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors can be obtained from Lonza Biologies, Inc. (Portsmouth, NH). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are herein incorporated by reference.
• the present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses ..host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g, promoter and/or enhancer) using techniques known of in the art.
• the host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g, a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a- prokaryotic cell, such as a bacterial cell.
• a host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression ofthe genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing ofthe foreign protein expressed.
• nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking a recombinant vector.
• the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g, the coding sequence), and/or to include genetic material (e.g, heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous .polynucleotides.
• endogenous genetic material e.g, the coding sequence
• genetic material e.g, heterologous polynucleotide sequences
• heterologous control regions e.g, promoter and/or enhancer
• endogenous polynucleotide sequences via homologous recombination
• heterologous control regions e.g, promoter and/or enhancer
• endogenous polynucleotide sequences via homologous recombination
• Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
• HPLC high performance liquid chromatography
• Polypeptides of the present invention can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host- mediated processes.
• N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
• the yeast Pichia pastoris is used to express polypeptides of the invention in a eukaryotic system.
• Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source.
• a main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O 2 . This reaction is catalyzed by the enzyme alcohol oxidase.
• Pichia pastoris In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O 2 .
• alcohol oxidase genes are highly active.
• alcohol oxidase produced from the A 0X1 gene comprises up to approximately 30%o of the total soluble protein in Pichia pastoris. See Ellis, S.B, et al, Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P.J, et al, Yeast 5:167-77 (1989); Tschopp, J.F, et al, Nucl Acids Res. 15:3859-76 (1987).
• heterologous coding sequence such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the A 0X1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
• the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins and J. Cregg, eds. The Humana Press, Totowa, NJ, 1998.
• This expression vector allows - expression and secretion of a polypeptide of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e, leader) located upstream of a multiple cloning site.
• PHO alkaline phosphatase
• yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.
• high-level expression of a heterologous coding sequence such as, for example, a polynucleotide of the present invention
• a heterologous coding sequence such as, for example, a polynucleotide of the present invention
• an expression vector such as, for example, pGAPZ or pGAPZalpha
• the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g, coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides.
• endogenous genetic material e.g, coding sequence
• genetic material e.g., heterologous polynucleotide sequences
• heterologous control regions e.g, promoter and/or enhancer
• endogenous polynucleotide sequences via homologous recombination
• heterologous control regions e.g, promoter and/or enhancer
• endogenous polynucleotide sequences via homologous recombination
• polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g, see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co, N.Y, and Hunkapiller et al. Nature, 310:105-111 (1984)).
• a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer.
• nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence.
• Non-classical amino acids include, but are not limited to, to the D- isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4- aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be
• the invention encompasses polypeptides of the present invention which are differentially modified during or after translation, e.g, by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8. protease, NaBH ; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
• Additional post-translational modifications encompassed by the invention include, for example, e.g, N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
• the polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and. isolation of the protein. ⁇ -
• suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-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.
• a luminescent material includes luminol
• bioluminescent materials include luciferase, luciferin, and aequorin
• suitable radioactive material include iodine ( 121 I, 123 I, 125 I, 131 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( m In, 112 In, ⁇ 3m In, 115m In), technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh,
• a polypeptide of the present invention or fragment or variant thereof is attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, 177 Lu, 90 Y, 166 Ho, and 153 Sm, to polypeptides.
• the radiometal ion associated with the macrocyclic chelators is U1 ln.
• the radiometal ion associated with the macrocyclic chelator is 90 Y.
• the macrocyclic chelator is 1,4,7,10- tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA).
• DOTA is attached to an antibody of the invention or fragment thereof via a linker molecule.
• linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art - see, for example, DeNardo et al, Clin Cancer Res. 4(10):2483-90 (1998); Peterson et al, Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby incorporated by reference in their entirety.
• the proteins of the invention may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide.
• Polypeptides of the invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
• Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and.
• the chemical moieties for derivitization may be selected l from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
• the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
• the polymer may be of any molecular weight, and may be branched or unbranched.
• the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
• Other sizes may be used, depending on the desired therapeutic profile (e.g, the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
• the polyethylene glycol may have an average molecular weight of about 200, 500,' 1000, 1500, 2000, 2500; 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
• the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575; Morpurgo et al, Appl Biochem. Biotechnol. 56:59-11 (1996); Vorobjev et al, Nucleosides Nucleotides 18:1145-1150 (1999); and Caliceti et al, Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference. [202] The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein.
• polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group.
• Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino.
• acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue.
• Sulfhydryl groups may also be used as a reactive group for . attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
• polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues.
• polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues.
• reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g, lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the. protein or to more than one type of amino acid residue (e.g, lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) ofthe protein.
• specific amino acid residues e.g, lysine, histidine, aspartic acid, glutamic acid, or cysteine
• One may specifically desire proteins ' chemically modified at the N-terminus.
• polyethylene glycol as an illustration ofthe present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
• the method of obtaining the N-terminally pegylated preparation i.e, separating this moiety from other monopegylated moieties if necessary
• Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
• pegylation of the proteins of the invention may be accomplished by any number of means.
• polyethylene glycol may be attached to the protein either directly or by an intervening linker.
• Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al, Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al. Intern. J. of Hematol. 68:1-18 (1998); U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.
• One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO 2 CH 2 CF 3 ).
• MPEG monmethoxy polyethylene glycol
• ClSO 2 CH 2 CF 3 tresylchloride
• polyethylene glycol is directly attached to amine groups of the protein.
• the invention includes protein- polyethylene glycol conjugates produced by reacting proteins " of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
• Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S.
• Patent No. 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins.
• Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with l,r-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p- - nitrophenolcarbonate, and various MPEG-succinate derivatives.
• a number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are ⁇ described in International Publication No. WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the • invention.
• the number of polyethylene glycol moieties attached to each protein of the invention may also vary.
• the pegylated proteins of the invention may be linked, on average, to 1, 2, ' 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules.
• the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13- 15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al, Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
• polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC”) is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
• HPLC high performance liquid chromatography
• the polypeptides of the invention may be in monomers or multimers (i.e, dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them.
• the polypeptides of the invention are monomers, dimers, trimers or tetramers!
• the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
• Multimers encompassed by the invention may be homomers or heteromers.
• the term homomer refers to a multimer containing only polypeptides corresponding to a protein ofthe invention (e.g, the amino acid sequence of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID NO:X or the complement of SEQ ID NO:X, the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or an amino acid sequence encoded by cDNA contained in Clone ID NO:Z (including fragments, variants, splice variants, and fusion proteins, corresponding to these as. described herein)).
• These homomers may contain polypeptides having identical or different amino acid sequences.
• a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g, containing two polypeptides having identical or different amino acid sequences) or a homotrimer (e.g, containing three polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
• heteromer refers to a multimer containing one or more heterologous polypeptides (i.e, polypeptides of different proteins) in addition to the polypeptides ofthe invention.
• the multimer ofthe invention is a heterodimer, a heterotrimer, or a heterotetramer.
• the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
• Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or' covalent associations and/or may be indirectly linked by, for example, liposome formation.
• multimers of the invention such as, for example, homodimers or homotrimers
• heteromultimers o the invention such as, for example, heterotrimers or heterotetramers
• polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protem ofthe invention) in solution.
• multimers of the invention are formed by covalent associations with and/or between, the polypeptides of the invention.
• covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g, that recited in SEQ ID NO:Y, encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or encoded by the cDNA contained in Clone ID NO:Z).
• the covalent associations are- cross-linking between cysteine residues located ithin the polypeptide sequences which interact in the native (i.e, naturally occurring) polypeptide.
• the covalent associations are the consequence of chemical or recombinant manipulation.
• covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein.
• covalent associations ' are between the heterologous sequence contained in a fusion protein of the invention (see, e.g, US Patent Number 5,478,925).
• the covalent associations are between the heterologous sequence contained in a Fc fusion protein ofthe invention (as described herein).
• covalent associations of fusion proteins, of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, osteoprotegerin (see, e.g.
• polypeptide linkers examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
• Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found.
• Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al. Science 240:1759, (1988)), and have since been found in a variety of different proteins.
• leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
• leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference.
• Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.
• Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity.
• Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers.
• a leucine zipper derived from lung surfactant protein D SPD
• SPD lung surfactant protein D
• Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides ofthe invention.
• proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence.
• proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins ofthe invention and anti-Flag® antibody.
• the multimers of the invention may be generated using chemical techniques known in the art.
• polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g, US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• linker molecules and linker molecule length optimization techniques known in the art
• multimers of the invention may be generated using techniques known in the art to form one or more inter- molecule cross-links between the cysteine -residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g, US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C- terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g, US Patent Number 5,478,925, which is herein incorporated by reference hi its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g, US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• multimers of the invention may be generated using genetic engineering techniques known in the art.
• polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N- terminus (lacking the leader sequence) (see, e.g, US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g, US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of the invention (e.g., a polypeptide or fragment or variant of the amino acid sequence of SEQ ID NO:Y or a polypeptide encoded by the cDNA contained in Clone ID. No:Z, and/or an epitope, of the present invention) as determined by immunoassays well known in the art for assaying specific antibody-antigen binding.
• TCR T-cell antigen receptors
• Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g, anti-Id antibodies to antibodies ofthe invention), intracellularly- made antibodies (i.e, intrabodies), and epitope-binding fragments of any ofthe above.
• antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e, molecules that contain an antigen binding site that immunospecifically binds an antigen.
• the immunoglobulin molecules of the invention can be of any type (e.g, IgG, IgE, IgM, IgD, IgA and IgY), class (e.g, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
• the immunoglobulin molecules of the invention are IgGl.
• the immunoglobulin molecules ofthe invention are IgG4.
• the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-, chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
• Antigen-binding antibpdy_ . fragments, including single-chain antibodies may comprise the variable region(s) alone or in combination with the entirety or a portion ofthe following: hinge region, CHI, CH2, and CH3 domains.
• antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains.
• the antibodies of the invention may be from any animal origin including birds and mammals.
• the antibodies are human, murine (e.g, mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.
• "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic " for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No.
• the antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al, J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al, J. Immunol. 148:1547-1553 (1992).
• Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind.
• the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g, by N-terminal and C-terminal positions, or by size in contiguous amino acid residues, or listed in the Tables and Figures.
• Preferred epitopes of the invention include the predicted epitopes shown in column 7 of Table IA, as well as polynucleotides that encode these epitopes.
• Antibodies which specifically bind any epitope or polypeptide ofthe present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
• Antibodies ofthe present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%>, at least 80%>, at least 75%>, at least 70%>, at least 65%o, at least 60%>, at least 55%, and at least 50%> identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
• antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof.
• Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%o,. less than 60%>, less than 55%, and less than 50%o identity (as calculated using methods known in the art and described herein) to a polypeptide ofthe present invention are also included in the present invention.
• the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein.
• antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions as described herein.
• Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention.
• Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10 "2 M, 10 "2 M, 5 X 10 "3 M, 10 “3 M, 5 X 10 “ 4 M, 10- 4 M, 5 X 10 '5 M, 10 "5 M, 5 X 10 "6 M, 10 “6 M, 5 X 10 "7 M, 10 7 M, 5 X 10 "8 M, 10 "8 M,
• the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein.
• the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %,-at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
• Antibodies of the present invention may act . as agonists or antagonists of the polypeptides . of the present invention.
• the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.
• antibodies of the present invention bind an antigenic epitope disclosed herein, or -a portion thereof.
• the invention features both receptor-specific antibodies and ligand-specific antibodies.
• the invention also features _receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e, signaling) may be determined by techniques described herein or otherwise known in the art.
• receptor activation can be determined by detecting the phosphorylation (e.g, tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
• antibodies are provided that inhibit ligand activity or receptor activity by at least 95%>, at least 90%>, at least 85%, at least 80%>, at least 75%>, at least, 70%), at least 60%, or at least 50% ofthe activity in absence of the antibody.
• the invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor- ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
• neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.
• antibodies which activate the receptor may act as receptor agonists, i.e, potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor.
• the antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities ofthe peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art.
• Antibodies of the present invention may be used, for example, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic, methods.
• the antibodies have utility in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g, Harlow et al. Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); incorporated by reference herein in its entirety.
• the antibodies of the present invention may be used either alone or in combination with other compositions.
• the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and no ⁇ -covalent conjugations) to polypeptides or other compositions.
• antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides;, or toxins. See, e.g, PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No.
• the antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response.
• the antibody derivatives include antibodies that have been modified, e.g, by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
• the derivative may contain one or more non-classical amino acids.
• the antibodies of the present invention may be generated by any suitable method known in the art.
• Polyclonal antibodies to an antigen-of- interest can be produced by various procedures well known in the art.
• a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
• adjuvants may be used to increase the imrnunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette- Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art. ..
• Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
• monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al. Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al, in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y, 1981) (said references incorporated by reference in their entireties).
• the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
• the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
• mice can be immunized with a polypeptide of the invention or a cell expressing such peptide.
• an immune response e.g, antibodies specific for the antigen are detected in the. mouse serum
• the mouse spleen is harvested and splenocytes isolated.
• the splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution.
• hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention.
• Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
• the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide ofthe invention.
• Another well known- method for producing both polyclonal and monoclonal human B cell lines is transformation using Epstein Barr "Virus (EBV).
• EBV Epstein Barr "Virus
• Protocols for generating EBV-transformed B cell lines are commonly known in the art, such as, for example, the protocol outlined in Chapter 7.22 of Current Protocols in Immunology, Coligan et al, Eds, 1994, John Wiley & Sons, NY, which is hereby incorporated in its entirety by reference.
• the source of B cells for transformation is commonly human peripheral blood, but B cells for transformation may also be derived from other sources including, but not limited to, lymph nodes, tonsil, spleen, tumor tissue, and infected tissues. Tissues are generally made into single cell suspensions prior to EBV transformation. Additionally, steps may be taken to either physically remove or inactivate T cells (e.g, by treatment with cyclosporin A) in B cell-containing samples, because T cells from individuals seropositive for anti-EBV antibodies can suppress B cell immortalization by EBV.
• EBV lines are generally polyclonal. However, over prolonged periods of cell cultures, EBV lines may become monoclonal or polyclonal as a result of the selective outgrowth of particular B cell clones.
• polyclonal EBV transformed lines may be subcloned (e.g., by limiting dilution culture) or fused with a suitable fusion partner and plated at limiting dilution to obtain monoclonal B cell lines.
• suitable fusion partners for EBV transformed cell lines include mouse myeloma cell lines (e.g, SP2/0, X63-Ag8.653), heteromyeloma cell lines, (human x mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g, GM 1500, SKO-007, RPMI 8226, and KR-4).
• the present invention also provides a method of generating polyclonal or monoclonal human antibodies against polypeptides ofthe invention or fragments thereof, comprising EBV-transformation of human B cells.
• Antibody fragments which recognize specific epitopes may be generated by known techniques.
• Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
• F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain ofthe heavy chain.
• the antibodies ofthe present invention can also be generated using various phage display methods known in the art.
• phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
• phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g, human or murine).
• Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g, using labeled antigen or antigen bound or captured to a solid surface or bead.
• Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
• Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al, J. Immunol. Methods 182:41-50 (1995); Ames et al, J. Immunol. Methods 184:177-186 (1995); Kettleborough et al, Eur. J. Immunol. 24:952-958 (1994); Persic et al. Gene 187 9-18 (1997); Burton et al.
• the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g, as described in detail below.
• techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al, BioTechniques 12(6):864-869 (1992); and Sawai et al, AJRI 34:26-34 (1995); and Better et al.
• a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
• Methods for producing "chimeric antibodies are known in the art. See e.g, Morrison, Science 229: 1202 (1985); Oi et al, BioTechniques 4:214 (1986); Gillies et al, (1989) J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety.
• Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule.
• CDRs complementarity determining regions
• framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
• These framework substitutions are identified by methods well known in the art, e.g, by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g. Queen et al, U.S. Patent No. 5,585,089; Riechmann et al.
• Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR- grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studn ' icka et al. Protein Engineering 7(6):805-814 (1994); Roguska. et al, PNAS 91:969-973 (1994)), and chain shuffling (U.S. Patent No. 5,565,332).
• Human antibodies are particularly desirable for therapeutic treatment of human patients.
• Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and 4,716.111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
• Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express
• the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
• the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
• the mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion ofthe JH region prevents endogenous antibody production.
• the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
• the chimeric mice are then bred to produce homozygous offspring which express human antibodies.
• the transgenic mice are immunized, in the normal fashion with a selected antigen, e.g, all or a portion of a polypeptide ofthe invention.
• Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
• the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
• IgE antibodies For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev.
• Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
• a selected non-human monoclonal antibody e.g, a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope.
• antibodies to the polypeptides ofthe invention can, in rum, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g, Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)).
• antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand.
• Such neutralizing anti- idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand(s)/receptor(s).
• anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and thereby block its biological activity.
• antibodies which bind to and enhance polypeptide multimerization and/or binding, and/or receptor/ligand multimerization, binding and/or signaling can be used to generate anti-idiotypes that function as agonists of a polypeptide of the invention and/or its ligand/receptor.
• Such agonistic anti-idiotypes or Fab fragments of such anti-idiotypes can. be used in therapeutic regimens as agonists of the polypeptides of the invention or its ligand(s)/receptor(s).
• anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and thereby promote or enhance its biological activity.
• Intrabodies of the invention can be produced using methods known in the art, such as those disclosed and reviewed in Chen et al. Hum. Gene Ther. 5:595-601 (1994); Marasco, W.A, Gene Ther. 4:11-15 (1997); Rondon and Marasco, Annu. Rev. Microbiol. 51:257-283 (1997); Proba et al, J. Mol. Biol. 275:245-253 (1998); Cohen et al, Oncogene 17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128 (1999); Ohage et al, J. Mol. Biol. 291:1129-1134 (1999); Wirtz and Steipe, Protein Sci. 8:2245-2250 (1999); Zhu et al, J. Immunol. Methods 231 :207-222 (1999); and references cited therein.
• the invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof.
• the invention also encompasses polynucleotides that hybridize under stringent or alternatively, under lower stringency hybridization conditions, e.g, as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide ofthe invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:Y, to a polypeptide encoded by a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or to a polypeptide encoded by the cDNA contained in Clone ID NO:Z.
• the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
• a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g, as described in Kutmeier et al, BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides- containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
• a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g, an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody ofthe invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g, a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may
• nucleotide sequence and corresponding amino acid sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g, recombinant DNA techniques, site directed mutagenesis, PCR, etc.
• the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g, by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
• CDRs complementarity determining regions
• one or more of the CDRs may be inserted within framework regions, e.g, into human framework regions to humanize a non-human antibody, as described supra.
• the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g, Chothia et al, J. Mol. Biol.
• the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
• one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
• Other alterations to the polynucleotide are encompassed by the present invention and within the skill ofthe art.
• a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g, humanized antibodies.
• the antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. Methods of producing antibodies include,, but are not limited to, hybridoma technology, EBV transformation, and other methods discussed herein as well as through the use recombinant DNA technology, as discussed below. [253] Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g, a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody.
• the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
• methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
• the invention thus, provides replicable vectors comprising' a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter.
• Such vectors may include the nucleotide sequence encoding the constant region ofthe antibody molecule (see, e.g, PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
• the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
• the invention includes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter.
• vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression ' of the entire immunoglobulin molecule, as detailed below.
• host-expression vector systems may be utilized to express the antibody molecules of the invention.
• Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ.
• These include but are not limited to microorganisms such as bacteria (e.g, E. coli, B.
• subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g, Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g, baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g, Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g, metallothionein promoter) or from mammalian .
• viruses e.g, the adenovirus late promoter; the vaccinia virus 7.5K promoter.
• bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
• mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al. Gene 45:101 (1986); Cockett et al, Bio/Technology 8:2 (1990)).
• a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
• vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, EMBO J.
• pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST).
• fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
• the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
• Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
• the virus grows in Spodoptera frugiperda cells.
• the antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
• a number of viral-based expression systems may be utilized.
• the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g, the late promoter and tripartite leader sequence.
• This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g, region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts, (e.g, see Logan & Shenk, Proc. Natl.
• Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of . origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al. Methods in Enzymol. 153:51-544 (1987)).
• a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g, cleavage) of protein products may be important for the function of the protein.
• Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
• eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
• Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, . and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
• cell lines which stably express the antibody molecule may be engineered.
• host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
• appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
• engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
• the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
• This method may advantageously be used to engineer cell lines which express the antibody molecule.
• Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
• a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al.
• hypoxanthine- guanine phosphoribosyltransferase Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)
• adenine phosphoribosyltransferase Lowy et al. Cell 22:817 (1980)
• antimetabolite resistance can be used as the basis of selection. for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al, Natl. Acad. Sci.
• Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively.
• An advantage of glutamine synthase based vectors are the availabilty of cell lines (e.g., the murine myeloma cell line, NS0) which are glutamine synthase negative.
• Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene.
• glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657 which are incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors that may be used according to the present invention are commercially available from suplliers, including, for example Lonza Biologies, Inc. (Portsmouth, NH): Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al, Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog.
• the host cell may be co-transfected with two expression vectors ofthe invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
• the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
• a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)).
• the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA. ' .
• an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g, ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
• chromatography e.g, ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
• centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
• the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
• the present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins.
• the fusion does not necessarily need to be direct, but may occur through linker sequences.
• the antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention.
• antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by rasing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.
• Antibodies fused or conjugated to the polypeptides ofthe present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al, supra, and .PCT publication WO 93/21232; EP 439,095; Naramura ' et al, Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981; Gillies et al, PNAS 89: 1428-1432 (1992); Fell et al, J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.
• the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
• the polypeptides ofthe present invention may be fused or conjugated to an antibody Fc region, or portion thereof.
• the antibody portion fused to a polypeptide ofthe present invention may comprise the constant region, hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.
• the polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
• Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions.
• Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM.
• Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g, U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al, Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al, J. Immunol.
• polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to facilitate purification.
• polypeptides of the present invention fused or conjugated to an antibody having disulfide- linked dimeric structures may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. See, for example, Fountoulakis et al, J. Biochem. 270:3958-3964, (1995).
• the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. See, for example, EP A 232,262.
• deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
• the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
• human proteins, such as hIL-5 have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al, J. Molecular Recognition 8:52- 58 (1995); Johanson et al, J. Biol. Chem. 270:9459-9471 (1995)).
• the antibodies or fragments .thereof of the present invention can be fused to marker sequences, ' such as a peptide to facilitate purification.
• the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc, 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
• hexa-histidine provides for convenient purification of the fusion protein.
• peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al. Cell 37:767 (1984)) and the ⁇ 'flag" tag.
• the present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent.
• the antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
• detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions.
• the detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
• suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
• suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
• suitable fluorescent materials include umbelliferone ' , fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
• an example ' of a luminescent material includes luminol;
• examples of bioluminescent materials include luciferase,, luciferin, and aequorin;
• suitable radioactive material include 1251, 1311, l l lln or 99Tc.
• an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g, a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g, alpha-emitters such as, for example, 213Bi.
• a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, .
• ethidium bromide emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
• Therapeutic agents include, but are not limited to, antimetabolites (e.g, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g, mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, ' busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g, dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g, vincris
• the conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
• the drug moiety may be a protein or polypeptide possessing a desired biological activity.
• Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g, TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al, Int.
• a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
• a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apop
• VEGI See, International Publication No. WO 99/23105
• a thrombotic agent or an anti- angiogenic agent e.g, angiostatin or endostatin
• biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
• IL-1 interleukin-1
• IL-2 interleukin-2
• IL-6 interleukin-6
• GM-CSF granulocyte macrophage colony stimulating factor
• G-CSF granulocyte colony stimulating factor
• Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
• Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polysty
• an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
• An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
• the antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. Translation products of the gene of the present invention may be useful as cell-specific markers, or more specifically as cellular markers that are differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, "panning" with antibody attached to a solid matrix (i.e, plate), and flow cytometry (See, e.g, U.S.
• MRD minimal residual disease
• GVHD Graft-versus- Host Disease
• these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
• the antibodies of the invention may be assayed for immunospecific binding by any method known in the art.
• the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, . radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name but a few.
• Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIP A buffer (l%o NP-40 or Triton X- 100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g, EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
• a lysis buffer such as RIP A buffer (l%o NP-40 or Triton X- 100,
• the ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g, western blot analysis.
• One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g, pre- clearing the cell lysate with sepharose beads).
• immunoprecipitation protocols see, e.g, Ausubel et al, eds, (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc, New York, section 10.16.1.
• Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g, 8%>- 20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g, PBS with 3%> BSA or non-fat milk), washing the membrane in washing buffer (e.g, PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g, an anti-human antibody) conjugated to an enzymatic substrate (e.g, horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g, 32P or 1251) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen.
• ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g, horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence ofthe antigen.
• a detectable compound such as an enzymatic substrate (e.g, horseradish peroxidase or alkaline phosphatase)
• a detectable compound such as an enzymatic substrate (e.g, horseradish peroxidase or alkaline phosphatase)
• a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
• ELISAs see, e.g, Ausubel et al, eds, (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc, New York, section 11.2.1.
• the binding affinity of an antibody to an antigen and the off-rate of an antibody- antigen interaction can be determined by competitive binding assays.
• a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g, 3H or 1251) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
• labeled antigen e.g, 3H or 1251
• the affinity ofthe antibody of interest for a particular antigen and the binding off- rates can be determined from the data by scatchard plot analysis.
• Competition with a second antibody can also be determined using radioimmunoassays.
• the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g, 3H or 1251) in the presence of increasing amounts of an unlabeled second antibody.
• Antibodies of the invention may be characterized using immunocytochemisty methods on cells (e.g, mammalian cells, such as CHO cells) transfected with a vector enabling the expression of an antigen or with vector alone using techniques commonly known in the art.
• cells e.g, mammalian cells, such as CHO cells
• Antibodies that bind antigen transfected cells, but not vector-only transfected cells, are antigen specific.
• the present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions.
• Therapeutic compounds of the invention include, but are not limited to, antibodies pf the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein).
• the antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide ofthe invention, including, but not limited to, any one or more of he diseases, disorders, or conditions described herein.
• the treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions.
• Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
• the present invention is directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more diseases, disorders, or conditions, including but not limited to: neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions, and/or as described elsewhere herein.
• diseases, disorders, or conditions including but not limited to: neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions, and/or as described elsewhere herein.
• Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (e.g, antibodies directed to the full length protein expressed on the cell surface of a mammalian cell; antibodies directed to an epitope of a polypeptide of the invention (such as, for example, a predicted linear epitope shown in column 7 of Table IA; or a conformational epitope, including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein).
• antibodies of the invention e.g, antibodies directed to the full length protein expressed on the cell surface of a mammalian cell
• antibodies directed to an epitope of a polypeptide of the invention such as, for example, a predicted linear epitope shown in column 7 of Table IA; or a conformational epitope, including fragments, analogs and derivatives thereof as described herein
• nucleic acids encoding antibodies of the invention (including fragments
• the antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein.
• the treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions.
• Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
• a summary of the ways in which the antibodies of the present invention may be used therapeutically includes bindmg polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below.
• the antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g, IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
• the antibodies of the invention may be administered alone or in combination with other types of treatments (e.g, radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred.
• human antibodies, fragments derivatives, analogs, or nucleic acids are administered to a human patient for therapy or prophylaxis.
• Preferred binding affinities m include those with a dissociation constant or Kd less than 5 X 10 "2 M, 10 "2 M, 5 X 10 "3 M, 10 “3 M, 5 X 10 "4 M, 10 "4 M, 5 X 10 "5 M, 10 “5 M, 5 X 10 '6 M, 10 “6 M, 5 X 10 "7 M, 10- 7 M, 5 X 10 "8 M, 10 “8 M, 5 X 10 "9 M, 10 "9 M, 5 X 10 "10 M, 10 “10 M, 5 X 10 "11 M, 10 "11 M, 5 X 10 "12 M, 10- 12 M, 5 X 10 "13 M, 10 " 13 M, 5 X 10 "14 M, 10 “14 M, 5 X 10 "15 M, and 10 "15 M.
• nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy.
• Gene therapy refers to therapy performed by the administration to a subject pf an expressed or expressible nucleic acid.
• the nucleic acids produce their encoded protein that mediates a therapeutic effect.
• the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host.
• nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being mducible or constitutive, and, optionally, tissue-specific.
• nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl.
• the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, ofthe antibody.
• Delivery ofthe nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
• the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g, by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g, by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Patent No.
• nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
• the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g, PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221).
• the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci.
• viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used.
• a retroviral vector can be used (see Miller et al, Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA.
• the nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient.
• retroviral vectors More detail about retroviral vectors can be found in Boesen et al, Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
• Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al, J r Clin. Invest. 93:644-651 (1994); Kiem et al. Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
• Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al.
• adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys.
• adenovirus vectors are used.
• Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al, Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146).
• Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. ' Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
• the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
• introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell- mediated gene transfer, spheroplast fusion, etc.
• Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g, Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al, Meth. Enzymol.
• the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
• the resulting recombinant cells can be delivered to a patient by various methods known in the art.
• Recombinant blood cells e.g, hematopoietic stem or progenitor cells
• the amount of cells envisioned for use depends on the desired effect, patient state, etc, and can be determined by one skilled in the art.
• Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells, such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g, as .obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
• the cell used for gene therapy is autologous to the patient.
• nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
• stem or progenitor cells are used.
• stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g.
• the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that- expression of the nucleic acid is controllable by the presence or absence of an appropriate inducer of transcription.
• the compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans.
• in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample.
• the effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays.
• in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
• the invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably a polypeptide or antibody of the invention.
• the compound- is substantially purified (e.g, substantially free from substances that limit its effect or produce undesired side-effects).
• the subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc, and is preferably a mammal, and most preferably human.
• Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
• Various delivery systems are known and can be used to administer a compound of the invention, e.g, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g, Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc.
• Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
• the compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g, oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
• Pulmonary administration can also be employed, e.g, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
• the pharmaceutical compounds or compositions of the invention may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g, in conjunction with a wound dressing after surgery, by injection, ' by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
• care must be taken to use materials to which the protein does not absorb.
• the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid, pp. 317-327; see generally ibid.)
• the compound or composition can be delivered in a controlled release system.
• a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al. Surgery 88:507 (1980); Saudek et al, N. Engl. J. Med. 321 :574 (1989)).
• polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres, Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J, Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al. Science 228:190 (1985); During et al, Ann. Neurol. 25:351 (1989); Howard et al, J.Neurosurg. 71:105 (1989)).
• a controlled release system can be placed in proximity ofthe therapeutic target, e.g, the brain, thus requiring only a fraction of the systemic dose (see, e.g, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
• the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g, by use of a retroviral vector (see U.S. Patent No.
• a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
• the present invention also provides pharmaceutical compositions.
• compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier.
• pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
• Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
• suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. ⁇
• These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
• composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
• Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
• Such compositions will contain a therapeutically effective amount ofthe compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
• the formulation should suit the mode of administration.
• the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
• compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
• the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
• the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
• composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
• an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
• the compounds of the invention can be formulated as neutral or salt forms.
• Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc, and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
• the amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges.
• Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
• the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
• the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg ofthe patient's body weight.
• human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
• the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g, into the brain) ofthe antibodies by modifications such as, for example, lipidation.
• the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
• a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
• Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the ag ⁇ ncy of manufacture, use or sale for human administration.
• Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention.
• the invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to. the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.
• the invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby . an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
• a diagnostic assay for diagnosing a disorder comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby . an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
• the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for
• Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g, see Jalkanen et al, J. Cell. Biol. 101:976-985 (1985); Jalkanen et al, J. Cell . Biol. 105:3087-3096 (1987)).
• Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
• Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• enzyme labels such as, glucose oxidase
• radioisotopes such as iodine (1251, 1211), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc)
• luminescent labels such as luminol
• fluorescent labels such as fluorescein and rhodamine, and biotin.
• diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is ' expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest.
• Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard
• the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
• the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc.
• the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein.
• In vivo tumor imaging is described in S.W. Burchiel et al, "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection pf Cancer, S.W. Burchiel and B. A. Rhodes, eds, Masson Publishing Inc. (1982)).
• the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
• monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
• Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used.
• CT computed tomography
• PET position emission tomography
• MRI magnetic resonance imaging
• sonography sonography
• the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al, U.S. Patent No. 5,441,050).
• the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument.
• the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (M I). '
• kits that can be used in the above methods.
• a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers.
• the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit.
• the kits ofthe present invention further comprise a control antibody which does not react with the polypeptide of interest.
• kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g, the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
• the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest.
• Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g, the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.
• the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached.
• a kit may also include a non-attached reporter-labeled anti-human antibody.
• binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter- labeled antibody.
• the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention.
• the diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody.
• the antibody is attached to a solid support.
• the antibody may be a monoclonal antibody.
• the detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a- labeled, competing antigen.
• test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention.
• the reagent After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support.
• the reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined.
• the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).
• the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96- well plate or filter rnaterial.__These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine- group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
• Streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
• the invention provides an assay system or kit for carrying out this diagnostic method.
• the kit generally includes a support with surface- bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti- antigen antibody.
• the polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome, thus each polynucleotide of the present invention can routinely be used as a chromosome marker using techniques known in the art. Table IA, column 9 provides the chromosome location of some of the polynucleotides ofthe invention.
• sequences can be mapped to chromosomes by preparing PCR primers
• Primers can optionally be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to SEQ ID NO:X will yield an amplified fragment.
• somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments.
• Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, preselection by hybridization to construct chromosome specific-cDNA libraries, and computer mapping techniques (See, e.g, Shuler, Trends Biotechnol 16:456- 459 (1998) which is hereby incorporated by reference in its entirety).
• Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread.
• FISH fluorescence in situ hybridization
• This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred.
• Verma et al "Human Chromosomes: a Manual of Basic Techniques," Pergamon Press, New York (1988).
• the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes).
• the present invention also provides a method for chromosomal localization which involves (a) preparing PCR primers from the polynucleotide sequences in Table 1 A and/or Table 2 and SEQ ID NO:X and (b) screening somatic cell hybrids containing individual chromosomes.
• the polynucleotides of the present invention would likewise be useful for radiation hybrid mapping, HAPPY mapping, and long range restriction mapping.
• HAPPY mapping high range restriction mapping
• the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an individual and comparing the measured gene expression level with a standard level of polynucleotide expression level, • whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder. Additional non-limiting examples of diagnostic methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g. Example 12). [350] In still another embodiment, the invention includes a kit for analyzing samples for the presence of proliferative and/or cancerous polynucleotides derived from a test subject.
• the kit includes at least one polynucleotide probe containing a nucleotide sequence that will specifically hybridize with a polynucleotide of the invention and a suitable container.
• the kit includes two polynucleotide probes defining an internal region of the polynucleotide of the invention, where each probe has one strand containing a 31'mer-end internal to the region.
• the probes may be useful as primers for polymerase chain reaction amplification.
• the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed polynucleotide ofthe invention expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
• measuring the expression level of polynucleotides of the invention is intended qualitatively or quantitatively measuring or estimating the level ofthe polypeptide of the invention or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g, by determining or estimating absolute protein level or mRNA level) or relatively (e.g, by comparing to the polypeptide level or mRNA level in a second biological, sample).
• the polypeptide level or mRNA level i the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the related disorder or being determined by averaging levels from a population of individuals not having a related disorder.
• a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
• biological sample any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains polypeptide of the present invention or the corresponding mRNA.
• biological samples include body fluids (such as semen, lymph, vaginal pool, sera, plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide of the present invention, and tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known ' in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
• the method(s) provided above may preferably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides of the invention are attached to a solid support.
• the support may be a "gene chip” or a "biological chip” as described in US Patents 5,837,832, 5,874,219, and 5,856,174.
• a gene chip with polynucleotides of the invention attached may be used to identify polymorphisms between the isolated polynucleotide sequences of the invention, with, polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e.
• the present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art.
• PNA peptide nucleic acids
• the use of PNAs would serve as the preferred form if the polynucleotides of the invention are incorporated onto a solid support, or gene chip.
• a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems).
• PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization.
• the compounds of the present invention have uses which include, but are not limited to, detecting cancer in mammals.
• the invention is useful during diagnosis of pathological cell proliferative neoplasias which include, but are not limited to: acute myelogenous leukemias including acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, . acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous leukemias including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc.
• Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.
• Pathological cell proliferative disorders are often associated with inappropriate activation of proto-oncogenes. (Gelmann, E. P. et al, "The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology," in Neoplastic Diseases of the Blood, Vol 1, Wiernik, P. H. et al. eds, 161-182 (1985)).
• Neoplasias are now believed to result from the qualitative alteration of a normal cellular gene product, or from the quantitative modification of gene expression by insertion into the chromosome of a viral sequence, by chromosomal translocation of a gene to a more actively transcribed region, or by some other mechanism.
• mutated or altered expression of specific genes is involved in the pathogenesis of some leukemias, among other tissues and cell types.
• the human counterparts of the oncogenes involved in some animal neoplasias have been amplified or translocated in some cases of human leukemia and carcinoma.
• c-myc expression is highly amplified in the non-lymphocytic leukemia cell line HL-60.
• HL-60 cells When HL-60 cells are chemically induced to stop proliferation, the level of c-myc is found to be downregulated.
• International Publication Number WO 91/15580 International Publication Number WO 91/15580.
• exposure of HL-60 cells to a DNA construct that is complementary to the 5' end of c-myc or c-myb blocks translation of the corresponding mRNAs which downregulates expression ofthe c-myc or c-myb proteins and causes arrest of cell proliferation and differentiation of the treated cells.
• International Publication Number WO 51/15580 Wickstrom et al, Proc. Natl. Acad. Sci.
• a polynucleotide of the present invention can be used to control gene expression through triple helix formation or through antisense DNA or RNA.
• Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Triple helix formation is discussed in, for instance Lee et al. Nucleic Acids Research 6: 3073 (1979); Cooney et al. Science 241: 456 (1988); and Dervan et al. Science 251 : 1360 (1991).
• polynucleotide Both methods rely on binding of the polynucleotide to a- complementary DNA or RNA.
• preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix - see Lee et al, Nucl. Acids Res. 3:173 (1979); Cooney et al. Science 241 :456 (1988); and Dervan et al. Science 251:1360 (1991)) or to the mRNA itself (antisense - Okano, J. Neurochem.
• oligonucleotide as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.
• the oligonucleotide described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of polypeptide of the present invention antigens.
• Non-limiting antisense and triple helix methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g, the section labeled "Antisense and Ribozyme (Antagonists)").
• Polynucleotides of the present invention are also useful in gene therapy.
• One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect.
• the polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner.
• Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. Additional non-limiting examples of gene therapy methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g, the sections labeled "Gene Therapy Methods", and Examples 16, 17 and 18).
• the polynucleotides are also useful for identifying individuals from minute biological samples.
• the United States military for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
• RFLP restriction fragment length polymorphism
• an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying . personnel.
• This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
• the polynucleotides of the present invention can be used as additional DNA markers for RFLP.
• the polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's, genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences.. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.
• DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g, blood, saliva, semen, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant, urine, fecal matter, etc, can be amplified using PCR.
• gene sequences amplified from polymorphic loci such as DQa class II HLA gene, are used in forensic biology to identify individuals. (Erlich, H, PCR Technology, Freeman and Co. (1992)).
• polynucleotides ofthe present invention can be used as polymorphic markers for forensic purposes.
• reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin.
• Appropriate reagents can comprise, for example, DNA probes or primers prepared from the sequences of the present invention, specific to tissues, including but not limited to those shown in Table 1 A. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination. Additional non-limiting examples of such uses are further described herein.
• polypeptides of the present invention are also useful as hybridization probes for differential identification of the tissue(s) or cell type(s) present in a biological sample.
• polypeptides and antibodies directed to polypeptides of the present invention are useful to provide imrnunological probes for differential identification of the tissue(s) (e.g, immunohistochemistry assays) or cell type(s) (e.g, immunocytochemistry assays).
• tissue expressing polypeptides and/or polynucleotides of the present invention may be detected in certain tissues (e.g, tissues expressing polypeptides and/or polynucleotides of the present invention, for example, those disclosed in column 8 of Table 1 A, and/or cancerous and/or wounded tissues) or bodily fluids (e.g, semen, lymph, vaginal pool, serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a "standard" gene expression level, i.e, the expression level in healthy tissue from an individual not having the disorder.
• tissues e.g, tissues expressing polypeptides and/or polynucleotides of the present invention, for example, those disclosed in column 8 of Table 1 A, and/or cancerous and/or wounded tissues
• bodily fluids e.g, semen, lymph, vaginal pool, serum, plasma, urine, synovial fluid or spinal fluid
• the invention provides a diagnostic method of a disorder, which involves: (a) assaying gene expression level in cells or body fluid of an individual ' ; (b) comparing the gene expression level with a standard gene expression, level, whereby an increase or decrease in the assayed gene expression level compared to the standard expression level is indicative of a disorder.
• the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to "subtract-out" known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a "gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.
• polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.
• Polypeptides and antibodies directed to polypeptides of the present invention are useful to provide imrnunological probes for differential identification of the tissue(s) (e.g, immunohistochemistry assays such as, for example, ABC immunoperoxidase (Hsu et al, J. Histochem. Cytochem. 29:577-580 (1981)) or cell type(s) (e.g, immunocytochemistry assays).
• Antibodies can be used to assay levels of polypeptides encoded by polynucleotides of the invention in a biological sample using classical immunohistological methods known to those of skill in the art (e.g, see Jalkanen, et al, J. Cell. Biol. 101:976- 985 (1985); Jalkanen, et al, J. Cell. Biol. 105:3087-3096 (1987)).
• Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
• ELISA enzyme linked immunosorbent assay
• RIA radioimmunoassay
• Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 131 I, 125 L 123 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115m In, 113m In, 112 In, m In), and technetium .( 99 Tc, 99m Tc), thallium ( 201 Ti), gallium ( 6s Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( I33 Xe), fluorine ( I8 F), 153 Sm, 177 Lu, l59 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhod
• proteins can also be detected in vivo by imaging.
• Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or - ESR.
• suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
• Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
• a protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (for example, 131 I, ⁇ 2 In, 99m Tc, ( 131 I, 125 I, 123 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115m In, n3ra In, ⁇ 2 In, m m), and technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F, 153 Sm, I77 Lu, ,59 Gd, 149 Pm, ' 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, I88 Re, 142 Pr, 105 Rh, 97 Ru), a radio-opaque substance,
• the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
• the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99m Tc.
• the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which express the polypeptide encoded by a polynucleotide of the invention.
• In vivo tumor imaging is described in S.W. Burchiel et al, "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: . The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds, Masson Publishing Inc. (1982)).
• the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (e.g, polypeptides encoded by polynucleotides of the invention and/or antibodies) that are associated with heterologous polypeptides or nucleic acids.
• polypeptides of the invention e.g, polypeptides encoded by polynucleotides of the invention and/or antibodies
• the invention provides a method for delivering a therapeutic protein into the targeted cell.
• the invention provides a method for delivering a single stranded nucleic acid (e.g, antisense or ribozymes) or double stranded nucleic acid (e.g, DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
• the invention provides a method for the specific destruction of cells (e.g, the destruction of tumor cells) by administering polypeptides of the invention in association with toxins or cytotoxic prodrugs
• toxin is meant one or more compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death.
• Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin.
• radioisotopes known in the art
• compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseu
• Toxin also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g, alpha-emitters such as, for example, 213 Bi, or other radioisotopes such as, for example, 103 Pd, 133 Xe, l31 I, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 35 S, 90 Y, ' 153 Sm, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, I I3 Sn, 90 Yttrium, I 17 Tin, 186 Rhenium, I66 Holmium, and 188 Rhenium; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• alpha-emitters such as, for example, 213 Bi
• radioisotopes such as, for example, 103 Pd, 133 Xe, l31 I,
• the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 90 Y.
• the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope ⁇ In.
• the invention provides a method for the specific destruction of cells (e.g, the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope 131 I.
• the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression level of a polypeptide of the present invention in cells or body fluid of an individual; and (b) comparing the assayed polypeptide expression level with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide expression level compared to the standard expression level is indicative of a disorder.
• a diagnostic method of a disorder involves (a) assaying the expression level of a polypeptide of the present invention in cells or body fluid of an individual; and (b) comparing the assayed polypeptide expression level with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide expression level compared to the standard expression level is indicative of a disorder.
• the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development ofthe disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
• polypeptides of the present invention can be used to treat or prevent diseases or conditions such as, for example, neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions.
• diseases or conditions such as, for example, neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions.
• patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels ofthe polypeptide (e.g, insulin), to supplement absent or decreased levels of a different polypeptide (e.g, hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide ' (e.g, an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g, by- binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g, soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g, blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).
• a polypeptide e.g, insulin
• a different polypeptide e.g, hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins
• a polypeptide ' e.g, an on
• antibodies directed to a polypeptide of the present invention can also be used to treat disease (as described supra, and elsewhere herein).
• administration of an antibody directed to a polypeptide of the present invention can bind, and/or neutralize the polypeptide, and/or reduce overproduction of the polypeptide.
• administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
• the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant. cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the biological activities described herein.
• the compounds of the present invention are useful for diagnosis, treatment, prevention and/or prognosis of various disorders in mammals, preferably humans.
• disorders include, but are not limited to, those described herein under the section heading "Biological Activities”.
• substantially altered (increased or decreased) levels of gene expression can. be detected in tissues, cells or bodily fluids (e.g, sera, plasma, urine, semen, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a "standard" gene expression level, that is, the expression level in tissues or bodily fluids from an individual not having the disorder.
• a diagnostic method useful during diagnosis of a disorder which involves measuring the expression level of the gene encoding the polypeptide in tissues, cells or body fluid from an individual and comparing the measured gene expression level with a standard ' gene expression level, whereby an increase or decrease in the gene expression level(s) compared to the standard is indicative of a disorder.
• diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.
• the present invention is also useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed gene expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
• a polypeptide of the invention may be used to diagnose and/or prognose diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1 A, column 8 (Tissue Distribution Library Code).
• tissue(s) in which the polypeptide of the invention including one, two, three, four, five, or more tissues disclosed in Table 1 A, column 8 (Tissue Distribution Library Code).
• the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g, by determining or estimating absolute protein level or mRNA level) or relatively (e.g, by comparing to the polypeptide level or mRNA level in a second biological sample).
• the polypeptide expression level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder.
• a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
• biological sample any biological sample obtained from an individual, cell line, tissue culture, or other source containing polypeptides of the invention (including portions thereof) or mRNA.
• biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) and tissue sources found to express the full length or fragments, thereof of a polypeptide or mRNA. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
• Total cellular RNA can be isolated from a biological sample vising any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the polypeptides of the invention are then assayed using any appropriate method. These include Northern blot analysis, SI nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR). [388] The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for.
• a diagnostic assay in accordance with the invention for detecting over-expression of polypeptides of the invention compared to normal control tissue samples may be used to detect the presence of tumors.
• Assay techniques that can be used to determine levels of a polypeptide, such as a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Assaying polypeptide levels in a biological sample can occur using any art-known method.
• Assaying polypeptide levels in a biological sample can occur using antibody-based techniques. For example, polypeptide expression in tissues can be studied with classical immunohistological methods (Jalkanen et al, J. Cell. Biol. 101 :976-985 (1985); Jalkanen, M, et al, J. Cell . Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting polypeptide gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
• ELISA enzyme linked immunosorbent assay
• RIA radioimmunoassay
• Suitable antibody assay labels include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 1 I2 In), and technetium ( 99m Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• enzyme labels such as, glucose oxidase, and radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 1 I2 In), and technetium ( 99m Tc)
• fluorescent labels such as fluorescein and rhodamine, and biotin.
• the tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the gene of inteest (such as, for example, cancer).
• the protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D, 1988, “Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which is incorporated herein by reference in its entirety.
• the isolated cells can be derived from cell culture or from a patient.
• the analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression ofthe gene.
• antibodies, or fragments of antibodies, such as those described herein may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
• antibodies, or fragments of antibodies directed to any one or all of the predicted epitope domains of the polypeptides of the invention may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
• antibodies, or fragments of antibodies directed to a conformational epitope of a polypeptide of the invention may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
• the antibodies (or fragments thereof), and/or polypeptides of the present invention may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of gene products or conserved variants or peptide fragments thereof.
• In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or polypeptide of the present invention.
• the antibody (or fragment thereof) or polypeptide is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
• Immunoassays and non-immunoassays for gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of binding gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
• the biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles -or soluble proteins.
• the support may then be washed with suitable buffers followed by treatment with the detectably labeled antibody or detectable polypeptide of the invention.
• the solid phase support may then be washed with the buffer a second time to remove unbound antibody or polypeptide.
• the antibody is subsequently labeled.
• the amount of bound label on solid support may then be detected by conventional means.
• solid phase support or carrier any support capable of binding an antigen or an antibody.
• supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
• the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
• the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
• the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
• the surface may be flat such as a sheet, test strip, etc.
• Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
• binding activity of a given lot of antibody or antigen polypeptide may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
• polypeptide or polynucleotide can also be detected in vivo by imaging.
• polypeptides and/or antibodies of the invention are used to image diseased cells, such as neoplasms.
• polynucleotides of the invention e.g, polynucleotides complementary to all or a portion of an mRNA
• antibodies e.g., antibodies directed to any one or a combination of the epitopes of a polypeptide of the invention, antibodies directed to a conformational epitope of a polypeptide of the invention, or antibodies directed to the full length polypeptide expressed on the cell surface of a mammalian cell
• Antibody labels or markers for in vivo imaging of polypeptides of the invention include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR.
• suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
• suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
• Such antibodies can be produced using techniques described herein or otherwise known in the art. For example methods for producing chimeric antibodies are known in the art. See, for- review, Morrison, Science 119: 1101 (1985); Oi et al, BioTechniques 4:114 (1986); Cabilly et al, U.S. Patent No. 4,816,567; Taniguchi et al, EP 171496; Morrison et al, EP 173494; Neuberger et al, WO 8601533; Robinson et al, WO 8702671; Boulianne et al, Nature 311:643 (1984); Neuberger et al, Nature 314:168 (1985).
• any polypeptides of the invention whose presence can be detected can be administered.
• polypeptides of the invention labeled with a radio- opaque or other appropriate compound can be administered and visualized in vivo, as discussed, above for labeled antibodies. Further, such polypeptides can be utilized for in vitro diagnostic procedures.
• a polypeptide-specific antibody or antibody fragment which has been labeled with an appropriate detectable ir ⁇ aging moiety such as a radioisotope (for example, 131 I, U2 In, 99 Tc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for a disorder.
• a radioisotope for example, 131 I, U2 In, 99 Tc
• a radio-opaque substance for example, a radio-opaque substance, or a material detectable by nuclear magnetic resonance
• the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
• the quantity of radioactivity injected will normally range from about 5 ..to 20 millicuries of 99m Tc.
• the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the antigenic protein.
• In vivo tumor imaging is described in S.W. Burchiel et al, "Immunopharmacokinetics of Radiolabeled .Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds, Masson Publishing Inc. (1982)). ' '
• an antibody of the present invention can be detectably labeled is by linking the same to a reporter enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A, "The Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller et al, J. Clin. Pathol. 31 :507- 520 (1978); Butler, J.E, Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL,; Ishikawa, E.
• EIA enzyme immunoassay
• the reporter enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
• Reporter enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and ' acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the reporter enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
• Detection may also be accomplished using any of a variety of other immunoassays.
• a radioimmunoassay RIA
• the radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
• fluorescent labeling compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
• the antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
• DTP A diethylenetriaminepentacetic acid
• EDTA ethylenediaminetetraacetic acid
• the antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
• the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
• chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
• a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
• Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
• a disease may be detected in a patient based on the presence of one or more proteins ofthe invention and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, urine, and/or tumor biopsies) obtained from the patient.
• a biological sample for example, blood, sera, urine, and/or tumor biopsies
• proteins may be used as markers to indicate the presence or absence of a disease or disorder, including cancer and/or as described elsewhere herein.
• proteins may be useful for the detection of other diseases and cancers.
• the bindmg agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample.
• Polynucleotide primers and probes may be used to detect the level of mRNA encoding polypeptides of the invention, which is also indicative of the presence or absence of a disease or disorder, including cancer.
• polypeptides of the invention should be present at a level that is at least three fold higher in diseased tissue than in normal tissue.
• the presence or absence of a disease in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value.
• the assay involves the use of a binding agent(s) immobilized on a solid support to bind to and remove the polypeptide of the invention from the remainder ofthe sample.
• the bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds , to the binding agent/polypeptide complex.
• detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin.
• a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample.
• Suitable polypeptides for use within such assays include polypeptides of the invention and portions thereof, or ' antibodies, to which the binding agent binds, as described above.
• the solid support may be any material known to those of skill in the art to which polypeptides of the invention may be attached.
• the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.
• the support may be a bead or disc, such as glass fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride.
• the support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Patent No. 5,359,681.
• the binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature.
• immobilization refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for the suitable _ amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day.
• contacting a well of plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 ug, and preferably about 100 ng to about 1 ug, is sufficient to immobilize an adequate amount of binding agent.
• plastic microtiter plate such as polystyrene or polyvinylchloride
• Covalent attachment of binding agent to a solid support may generally be. achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent.
• a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent.
• the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g. Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
• gene therapy methods for treating or preventing disorders, diseases and conditions.
• the gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of the polypeptide of the present invention.
• This method requires a polynucleotide which codes for a polypeptide ofthe present invention operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue.
• Such gene' therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.
• cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the present invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide ofthe present invention.
• a polynucleotide DNA or RNA
• Such methods are well-known in the art. For example, see Belldegrun, A, et al, J. Natl. Cancer Inst. 85: 207-2-16 (1993); Ferrantini, M. et al. Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al, J.
• the cells which are engineered are arterial, cells.
• the arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.
• the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like).
• the polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
• the polynucleotide of the present invention is delivered as a naked polynucleotide.
• naked polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like.
• the polynucleotide of the present invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.
• the polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication.
• Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; pSVK3, pBPV, ' pMSG and pSVL available from Pharmacia; and pEFl/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen.
• Other suitable, vectors will be readily apparent to the skilled artisan.
• Any strong promoter known to those skilled in the art can- be used for driving the expression of the polynucleotide sequence.
• Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters.
• CMV cytomegalovirus
• RSV respiratory syncytial virus
• inducible promoters such as the MMT promoter, the metallothionein promoter
• heat shock promoters such as the albumin promoter
• the ApoAI promoter the ApoAI promoter
• the promoter also may be the native promoter for the polynucleotide of the present invention.
• the promoter also may be the native promoter for the polynucleotide of the present invention.
• the ,polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue.
• Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma ofthe circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells.
• an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight.
• the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg.
• this dosage will vary according to the tissue site of injection.
• the appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.
• the preferred route of administration is by the parenteral route of injection into the interstitial space of tissues.
• parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes' of the nose.
• naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
• the naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called "gene guns". These delivery methods are known in the art.
• constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.
• the polynucleotide constructs are complexed in a liposome preparation.
• Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations.
• cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid.
• Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al, Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference); mRNA (Malone et al, Proc. Natl. Acad.
• Cationic liposomes are readily available.
• N[l-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et al, Proc. Natl Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference).
• Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).
• cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication No. WO 90/11092 (which is herein incorporated by reference) for a description of the synthesis of DOTAP (1,2- bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g, P. Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.
• anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials.
• Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), ..dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl. ethanolamine. (DOPE), among others.
• DOPC dioleoylphosphatidyl choline
• DOPG dioleoylphosphatidyl glycerol
• DOPE dioleoylphoshatidyl. ethanolamine.
• These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
• DOPC dioleoylphosphatidyl choline
• DOPG dioleoylphosphatidyl glycerol
• DOPE dioleoylphosphatidyl ethanolamine
• DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water.
• the sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC.
• negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size.
• Other methods are known and available to those of skill in the art.
• the liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred.
• MLVs multilamellar vesicles
• SUVs small unilamellar vesicles
• LUVs large unilamellar vesicles
• the various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g, Straubinger et al. Methods of Immunology (1983), 101:512-527, which is herein incorporated by reference.
• MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipi ' d on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated.
• SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes.
• the material to be entrapped is added to a suspension of preformed MLVs and then sonicated.
• liposomes containing cationic lipids the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA.
• the liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA.
• SUVs find use with small nucleic acid fragments.
• LUVs are prepared by a number of methods, well known in the art. Commonly used methods mclude Ca 2+ -EDTA chelation (Papahadjopoulos et al, Biochim. Biophys. Acta' (1975) 394:483; Wilson et al. Cell 17:77 (1979)); ether injection (Deamer, D. and Bangham, A, Biochim. Biophys. Acta 443:629 (1976); Ostro et al, Biochem. Biophys. Res. Commun. 76:836 (1977); Fraley et al, Proc. Natl. Acad. Sci. USA 76:3348 (1979)); detergent dialysis (Enoch, H. and Strittmatter, P, Proc.
• the ratio of DNA to liposomes will be from about 10:1 to about 1:10.
• the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3 : 1 to about 1:3. Still more preferably, the ratio will be about 1:1.
• U.S. Patent No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice.
• WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals.
• U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 provide methods for delivering DNA-cationic lipid complexes to mammals.
• cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding a polypeptide ofthe present invention.
• Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
• the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
• packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA 12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy 1 :5-14 (1990), which is incorporated herein by reference in its entirety.
• the vector may transduce the packaging cells through any means known in the art. Such means include ⁇ ut are not limited to, electroporation, the use of liposomes, and CaPO 4 precipitation.
• the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
• the producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a polypeptide of the present invention.
• retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
• the transduced eukaryotic cells will express a polypeptide ofthe present invention.
• cells are ' engineered, ex vivo or in vivo, with polynucleotide contained in an adenovirus vector.
• Adenovirus can be manipulated such that it encodes and expresses a polypeptide of the present invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz et al. Am. Rev. Respir. Dis.109:233-238 (1974)).
• adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha- l ' -antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434; Rosenfeld et al, (1992) Cell 68:143-155). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606). [438] Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet.
• the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the El region ofadeno virus and constitutively express Ela and Elb, which complement the defective adenoviruses by providing the products of the genes deleted from the vector.
• Ad2 other varieties of adenovirus (e.g, Ad3, Ad5, and Ad7) are also useful in the present invention.
• the adenoviruses used in the present invention are replication deficient.
• Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles.
• the resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells.
• Replication deficient adenoviruses may be deleted in one or more of all or a portion ofthe following genes: Ela, Elb, E3, E4, E2a, or LI through L5.
• the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV).
• AAV adeno-associated virus
• AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N, Curr. Topics in Microbiol. Immunol. 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Patent Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.
• an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration.
• the polynucleotide construct is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989).
• the recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc.
• Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses.
• the packaging cells Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo.
• the transduced cells will contain the polynucleotide construct integrated into its genome, and will express a pplypeptide ofthe invention.
• Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding a polypeptide of the present invention) via homologous recombination (see, e.g, U.S. Patent No. 5,641,670, issued June 24, 1997; International Publication No. WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller et al, Proc. Natl. ' Acad. Sci. USA 86:8932-8935. (198-9); and Zijlstra et al. Nature 342:435-438 (1989), which are herein encorporated by reference.
• This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.
• Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein.
• the targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence.
• the targeting sequence will be sufficiently near the 5' end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.
• the promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5' and 3' ends.
• the 3' end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5' end of the second targeting sequence contains the same restriction site as the 3' end of the amplified promoter.
• the amplified promoter and targeting sequences are digested and ligated together.
• the promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide; or in. conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc, described in more detail above.
• the P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.
• the promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression ofthe endogenous sequence.
• the polynucleotide encoding a polypeptide ofthe present invention may contain a secretory signal sequence that facilitates secretion of the protein.
• the signal sequence is positioned in the coding region ofthe polynucleotide to be expressed towards or at the 5' end of the coding region.
• the signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.
• any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in -the expression of one or more molecules in an amount sufficient to provide a therapeutic effect.
• This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (i.e, "gene guns"), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g, Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery.
• a preferred method of local administration is by direct injection.
• a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries.
• Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.
• compositions useful in systemic administration include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention.
• Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site.
• suitable delivery vehicles for use with systemic administration comprise liposomes comprising polypeptides of the invention for targeting the vehicle to a particular site.
• Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in, the art (see, for example, Stribling et al, Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein by reference).
• Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art.
• Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g, DMSO) that is capable of passing into the skin.
• a lipophilic reagent e.g, DMSO
• Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian.
• Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly preferred.
• Polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides or polypeptides, or agonists or antagonists of the present invention, do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides and polypeptides, and agonists or. antagonists could be used to treat the associated disease.
• Endocrine-associated proteins are believed to be involved in biological activities associated with diseases and/or disorders of the endocrine system and/or diseases and/or disorders related to hormone imbalance.
• compositions of the invention may be used in the diagnosis, prognosis, prevention, and/or treatment of diseases and/or disorders associated with aberrant endocrine system, hormone and/or homone antagonist related activity.
• compositions of the invention may be used in the diagnosis, prognosis, prevention, and/or treatment of diseases and/or disorders relating to endocrine disorders (e.g., disorders of the pancreas, disorders of the pituitary gland, disorders of the adrenal glands, disorders ofthe hypothalamus, disorders of the thyroid gland, disorders of the parathyroid gland, disorders of the ovaries, disorders of the testes, and/or disorders of .
• diseases and/or disorders relating to endocrine disorders e.g., disorders of the pancreas, disorders of the pituitary gland, disorders of the adrenal glands, disorders ofthe hypothalamus, disorders of the thyroid gland, disorders of the parathyroid gland, disorders of the ovaries, disorders of the testes, and/or disorders of .
• neoplastic disorders e.g., pheochromocytoma, neuroblastoma, multiple endocrine neoplasia, cancers of endocrine tissues
• neoplastic disorders e.g., pheochromocytoma, neuroblastoma, multiple endocrine neoplasia, cancers of endocrine tissues
• compositions of the invention may be used in the diagnosis, prognosis, prevention, and/or treatment of reproductive disorders (e.g, infertility and impotence), menstrual disorders (e.g, premenstrual syndrome, dysmenorrhea, amenorrhea, menorrhagia, endometriosis), complications of pregnancy (e.g, miscarriage, preterm labor, ectopic pregnancy, and preeclampsia), and/or as described under the section entitled "Reproductive System Disorders” below.
• reproductive disorders e.g, infertility and impotence
• menstrual disorders e.g, premenstrual syndrome, dysmenorrhea, amenorrhea, menorrhagia, endometriosis
• complications of pregnancy e.g, miscarriage, preterm labor, ectopic pregnancy, and preeclampsia
• a polypeptide of the invention may be used to diagnose, prognose, prevent, and/or treat diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table IA, column 8 (Tissue Distribution Library Code).
• polynucleotides, translation products and antibodies of the invention are useful in the diagnosis, prognosis, prevention, and/or treatment of diseases and or disorders associated with activities that include, but are not limited to, hormone production, hormone regulation, hormone adsorption, hormone storage, hormone secretion, and/or responsiveness to hormones.
• polynucleotides, translation products and antibodies corresponding to this gene may be useful for the diagnosis, detection and/or treatment of diseases and/or disorders associated with the following systems.
• Polynucleotides or polypeptides, or agonists or antagonists of the present invention may be used to treat, prevent, diagnose, and/or prognose disorders and/or diseases related to hormone imbalance, and/or disorders or diseases ofthe endocrine system.
• Hormones secreted by the glands of the .endocrine system control physical growth, sexual function, metabolism, and other functions. Disorders may be classified in two ways: disturbances in the production of hormones, and the inability of tissues to respond to hormones. The etiology of these hormone imbalance or endocrine system diseases, disorders or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g, by chemotherapy, injury or toxins), or infectious.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention can be used as a marker or detector of a particular disease or disorder related to the endocrine system and/or hormone imbalance.
• Endocrine system and/or hormone imbalance and/or diseases encompass disorders of uterine motility including, but not limited to: complications with pregnancy and labor (e.g, pre-term labor, post-term pregnancy, spontaneous abortion, and slow or stopped labor); and disorders and/or diseases of the menstrual cycle (e.g, dysmenorrhea and endometriosis).
• complications with pregnancy and labor e.g, pre-term labor, post-term pregnancy, spontaneous abortion, and slow or stopped labor
• disorders and/or diseases of the menstrual cycle e.g, dysmenorrhea and endometriosis
• Endocrine system and/or hormone imbalance disorders and/or diseases include disorders and/or diseases of the pancreas, such as, for example, diabetes mellitus, diabetes insipidus, congenital pancreatic agenesis, pheochromocytoma— islet cell tumor syndrome; disorders and/or diseases of the adrenal glands such as, for example, Addison's Disease, corticosteroid deficiency, virilizing disease, hirsutism, Cushing's Syndrome, hyperaldosteronism, pheochromocytoma; disorders and/or diseases of the pituitary gland, such as, for example, hyperpituitarism, hypopituitarism, pituitary dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, gigantism; disorders and/or diseases of the thyroid, including but not limited to, hyperthyroidism, hypothyroidism, Plummer's disease, Grav
• endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases of the testes or- ovaries, including cancer.
• Other disorders and/or diseases of the testes or ovaries further include, for example, ovarian cancer, polycystic ovary syndrome, Klinefelter's syndrome, vanishing testes syndrome (bilateral anorchia), congenital absence of Leydig's cells, cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillary haemangioma of the testis (benign), neoplasias of the testis and neo-testis.
• endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases such as, for example, polyglandular deficiency syndromes, pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and disorders and/or cancers of endocrine tissues.
• a polypeptide of the invention may be used to diagnose, prognose, prevent, and/or treat endocrine diseases and/or disorders associated with the tissue(s) in which the polypeptide ofthe invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table IA, column 8 (Tissue Distribution Library Code).
• the polynucleotides or polypeptides, or agonists or antagonists of the invention may be used for the diagnosis, treatment, or prevention of diseases and/or disorders of the reproductive system.
• Reproductive system disorders that can be treated by the compositions of the invention, include, but are not limited to, reproductive system injuries, infections, neoplastic disorders, congenital defects, and diseases or disorders which result in infertility, complications with pregnancy, labor, or parturition, and postpartum difficulties.
• Reproductive system disorders and/or diseases include diseases and/or disorders of the testes, including testicular atrophy, testicular feminization, cryptorchism (unilateral and bilateral), anorchia, ectopic testis, epididymitis and orchitis (typically resulting from infections such as, for example, gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion, vasitis nodosa, germ cell tumors (e.g, seminomas, embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk sac tumors, and teratomas), stromal tumors (e.g., Leydig cell tumors), hydrocele, hematocele, varicocele, spermatocele, inguinal hernia, and disorders of sperm production (e.g, immotile cilia syndrome, aspermia, asthenozo
• Reproductive system disorders also include disorders of the prostate gland, such as acute non-bacterial prostatitis, chronic non-bacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, prostatodystonia, prostatosis, granulomatous prostatitis, malacoplakia, benign prostatic hypertrophy or hyperplasia, and prostate neoplastic disorders, including adenocarcinomas, transitional cell carcinomas, ductal carcinomas, and squamous cell carcinomas.
• the compositions of the invention may .
• disorders or diseases of the penis and urethra including inflammatory disorders, such as balanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea, non-gonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome, condyloma acuminatum, condyloma latum, and pearly penile papules; urethral abnormalities, such as hypospadias, epispadias, and phimosis; premalignant lesions, including Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis, giant condyloma of Buscke-Lowenstein, and varrucous carcinoma; penile cancers, including squa
• diseases and/or disorders of the vas deferens include vasculititis and CBAVD (congenital bilateral absence of the vas deferens); additionally, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the diagnosis, treatment, and/or prevention of diseases and/or disorders of the seminal vesicles, including hydatid disease, congenital chloride diarrhea, and polycystic kidney disease.
• disorders and/or diseases of the male reproductive system include, for example, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, high fever, multiple sclerosis, and gynecomastia.
• the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the diagnosis, treatment, and/or prevention of diseases and/or disorders of the vagina and vulva, including bacterial vaginosis, Candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale, lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma, vulvar trauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonas vaginitis, condyloma acuminatum, syphilis, niolluscum contagiosum, atrophic vaginitis, Paget's disease, lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome, vaginismus, vulvovaginit
• disorders and/or diseases of the uterus include dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding (e.g, due to aberrant hormonal signals), and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, and sarcomas.
• polypeptides, polynucleotides, or agonists or antagonists of the invention may be useful as a marker or detector of, as well as in the diagnosis, treatment, and/or prevention of congenital uterine abnormalities, such as bicornuate uterus, septate uterus, simple unicornuate uterus, unicornuate uterus with a noncavitary rudimentary horn, unicornuate uterus with a non-communicating cavitary rudimentary horn, unicornuate uterus with a communicating cavitary horn, arcuate uterus, uterine didelfus, and T-shaped . uterus.
• congenital uterine abnormalities such as bicornuate uterus, septate uterus, simple unicornuate uterus, unicornuate uterus with a noncavitary rudimentary horn, unicornuate uterus with a non-communicating cavitary rudimentary horn, unicornuate uterus with
• Ovarian diseases and/or disorders include anovulation, polycystic ovary syndrome (Stein-Leventhal syndrome), ovarian cysts, ovarian hypofunction, ovarian insensitivity to gonadotfopins, ovarian overproduction of androgens, right ovarian vein syndrome, amenorrhea, hirutism, and ovarian cancer (including, but not limited to, primary and secondary cancerous growth, Sertoli-Leydig tumors, endometriod carcinoma of the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma, and Ovarian Krukenberg tumors).
• Cervical diseases and/or disorders include cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, and cervical neoplasms (including, for example, cervical carcinoma, squamous metaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, and columnar cell neoplasia).
• diseases and/or disorders of the reproductive system include disorders and/or diseases of pregnancy, including miscarriage and stillbirth, such as early abortion, late abortion, spontaneous abortion, induced abortion, therapeutic abortion, threatened abortion, missed abortion, incomplete abortion, complete abortion, habitual abortion, missed abortion, and septic abortion; ectopic pregnancy, anemia, Rh incompatibility, vaginal bleeding during pregnancy, gestational diabetes, intrauterine growth retardation, polyhydramnios, HELLP syndrome, abruptio placentae, placenta previa, hyperemesis, preeclampsia, eclampsia, herpes gestationis, and urticaria of pregnancy.
• polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the diagnosis, treatment, and/or prevention of diseases that can complicate pregnancy, including heart disease, heart failure, rheumatic heart disease, congenital heart disease, mitral valve prolapse, high blood pressure, anemia, kidney disease, infectious disease (e.g, rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HIV, AIDS, and genital herpes), diabetes mellitus, Graves' disease, thyroiditis, hypothyroidism, Hashimoto's thyroiditis, chronic active hepatitis, cirrhosis of the liver, primary biliary cirrhosis, asthma, systemic lupus eryematosis, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian
• Complications associated with labor and parturition include premature rupture ofthe membranes, pre-term labor, post-term pregnancy, postmaturity, labor that progresses too slowly, fetal distress (e.g, abnormal heart rate (fetal or maternal), breathing problems ⁇ and abnormal fetal position), shoulder dystocia, prolapsed umbilical cord, amniotic fluid embolism, and aberrant uterine bleeding.
• fetal distress e.g, abnormal heart rate (fetal or maternal), breathing problems ⁇ and abnormal fetal position
• shoulder dystocia e.g, prolapsed umbilical cord, amniotic fluid embolism, and aberrant uterine bleeding.
• diseases and/or disorders of the postdelivery period including endometritis, myometritis, parametritis, peritonitis, pelvic thrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis, saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage, and inverted uterus.
• disorders and/or diseases of the female reproductive system that may be diagnosed, treated, and/or prevented by the polynucleotides, polypeptides, and agonists or antagonists of the present invention include, for example, Turner's syndrome, pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory disease, pelvic congestion (vascular engorgement), frigidity, anorgasmia, dyspareunia, ruptured fallopian tube,- and Mittel inconvenience.
• Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing and/or prognosing diseases, disorders, and/or conditions of the immune system, by, for example, activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells.
• Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
• immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g, by chemotherapy or toxins), or infectious.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.
• a polypeptide of the invention may be used to treat diseases and disorders of the immune system and/or to inhibit or enhance an immune response generated by cells associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1 A, column 8 (Tissue Distribution Library Code).
• Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing, and/or prognosing immunodeficiencies, including both congenital and acquired immunodeficiencies.
• B cell immunodeficiencies in which immunoglobulin levels B cell function and/or B cell numbers are decreased include: X-linked agammaglobulinemia (Bruton's disease), X-linked infantile agammaglobulinemia, X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, X-linked lymphoproliferative syndrome (XLP), agammaglobulinemia including congenital and acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, unspecified hypogammaglobulinemia, recessive agammaglobulinemia (Swiss type); Selective IgM deficiency, selective IgA deficiency, selective IgG subclass deficiencies, IgG subclass deficiency (with or without IgA deficiency), I
• Ataxia-telangiectasia or conditions associated with ataxia-telangiectasia- are treated, prevented, diagnosed, and/or prognosing using the polypeptides or polynucleotides ofthe invention, and/or agonists or antagonists thereof.
• Examples of congenital immunodeficiencies in which T cell and/or B cell function and/or number is decreased include, but are not limited to: DiGeorge anomaly, severe combined immunodeficiencies (SCID) (including, but not limited to, X-linked SCID, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich syndrome, and ataxia telangiectasia), thymic hypoplasia, third and fourth pharyngeal pouch syndrome, 22ql l.2 deletion, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4+ T-lymphocytopenia, immunodeficiency with predominant T cell defect (unspecified), and unspecified immunodeficiency of cell mediated immunity.
• SCID severe combined immunode
• DiGeorge anomaly or conditions associated with DiGeorge anomaly are treated, prevented, diagnosed, and or prognosed using polypeptides or polynucleotides ofthe invention, or antagonists or agonists thereof.
• Other immunodeficiencies that may be treated, prevented, diagnosed, and/or prognosed using polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof, include, but are not limited to, chronic granulomatous disease, Chediak- Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6-phosphate dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency, complement component deficiencies (including Cl, C2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic alymphoplasia-aplasia, immunodeficiency with th
• the immunodeficiencies and/or conditions associated with the immunodeficiencies recited above are treated, prevented, diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies, and or agonists or antagonists of the present invention.
• polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among immunodeficient individuals.
• polynucleotides, .polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among B cell and or T cell immunodeficient individuals.
• the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing and/or prognosing autoimmune disorders.
• Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction ofthe host tissue. Therefore, the administration of polynucleotides and polypeptides of the invention that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.
• Autoimmune diseases or disorders that may be treated, prevented, diagnosed and/or prognosed by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, , one or more of the following: systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia purpura, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, purpura (e.g, Henloch-Scoenlein purpura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), and insulin- resistant diabetes me
• Additional disorders that are likely to have an autoimmune component that may be treated, prevented, and/or diagnosed with the compositions of the invention include, but are not limited to, type II collagen-induced arthritis, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye disorders.
• Additional disorders that are likely to have an autoimmune component that may be treated, prevented,, diagnosed and/or prognosed with the compositions of the invention include, but are not limited to, scleroderma with anti-collagen antibodies (often characterized, e.g, by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g, by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis (often characterized, e.g, by nonhistone ANA), pernicious anemia (often characterized, e.g, by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g, by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g, by antispermatozoal antibodies), glomerulonephritis (often characterized, e.g, by glomerular basement membrane
• Additional disorders that may have an autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the compositions of the invention include, but are not limited to, chronic active hepatitis (often characterized, e.g, by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g, by mitochondria antibodies), other endocrine gland failure (often characterized, e.g, by specific tissue antibodies in some cases), vitiligo (often characterized, e.g, by melanocyte antibodies), vasculitis (often characterized, e.g, by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g, by myocardial antibodies), cardiotomy syndrome (often characterized, e.g, by myocardial antibodies), urticaria (often characterized, .
• chronic active hepatitis often characterized, e.g, by smooth muscle antibodies
• primary biliary cirrhosis
• IgG and IgM antibodies to IgE e.g, by IgG and IgM antibodies to IgE
• atopic dermatitis e.g., by IgG and IgM antibodies to IgE
• asthma e.g., by IgG and IgM antibodies to IgE
• many other inflammatory, granulomatous, degenerative, and atrophic disorders e.g, by IgG and IgM antibodies to IgE
• the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using for example, antagonists or agonists, polypeptides or polynucleotides, or antibodies of the present invention.
• rheumatoid arthritis is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists ofthe present invention.
• systemic lupus erythematosus is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
• idiopathic thrombocytopenia purpura is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.

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