JP2003526369A - Novel polynucleotides and nucleic acids encoding the same - Google Patents

Abstract

  (57) [Summary] The present invention provides novel isolated NOVX polynucleotides and polypeptides encoded by NOVX polynucleotides. Antibodies that immunospecifically bind to a NOVX polypeptide, or any derivative, variant, variant or fragment of a NOVX polypeptide, polynucleotide or antibody are also provided. The present invention further provides methods of utilizing NOVX polypeptides, polynucleotides and antibodies for the detection and treatment of a wide variety of pathological conditions, as well as other uses.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates generally to nucleic acids and polypeptides encoded thereby.

BACKGROUND OF THE INVENTION The present invention relates generally to nucleic acids and polypeptides encoded thereby. More specifically, the present invention relates to nucleic acids encoding cytoplasmic, nuclear, membrane-bound, and secreted polypeptides, and vectors, host cells, antibodies and recombinant methods for producing these nucleic acids and polypeptides. .

SUMMARY OF THE INVENTION The present invention is based, in part, on the discovery of novel polynucleotide sequences that encode novel polypeptides.

Accordingly, in one aspect, the invention features SEQ ID NOs: 1, 3, 5, 7, 9, 11.
, 13, 15, 17, 19 or 21 sequences, or fragments thereof,
Provided are isolated nucleic acid molecules including homologs, analogs or derivatives. This nucleic acid is, for example, SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20.
Alternatively, it may comprise a nucleic acid sequence encoding a polypeptide that is at least 85% identical to a polypeptide comprising 22 amino acid sequences. The nucleic acid can be, for example, a genomic DNA fragment, or a cDNA molecule.

Also included in the invention are vectors that include one or more of the nucleic acids described herein, and cells that include the vectors or nucleic acids described herein.

The present invention also relates to host cells transformed with a vector containing any of the above nucleic acid molecules.

In another aspect, the invention includes a pharmaceutical composition that includes a NOVX nucleic acid and a pharmaceutically acceptable carrier or diluent.

[0008] In a further aspect, the invention includes a substantially purified NOVX polypeptide (eg, a NOVX polypeptide encoded by a NOVX nucleic acid, and any fragment, homolog, analog, and derivative thereof). The present invention also includes pharmaceutical compositions that include the NOVX polypeptides and a pharmaceutically acceptable carrier or diluent.

In yet a further aspect, the invention provides an antibody that specifically binds to a NOVX polypeptide. The antibody can be, for example, a monoclonal or polyclonal antibody, and fragments, homologs, analogs, and derivatives thereof. The invention also includes pharmaceutical compositions that include the NOVX antibody and a pharmaceutically acceptable carrier or diluent. The invention also relates to an isolated antibody that binds to an epitope of a polypeptide encoded by any of the above nucleic acid molecules.

[0010]   The present invention also includes kits containing any of the above pharmaceutical compositions.

The invention further provides a cell containing a NOVX nucleic acid (eg, a vector containing a NOVX nucleic acid) and culturing the cell under conditions sufficient to express the NOVX polypeptide encoded by the nucleic acid. By providing a NOVX polypeptide. The expressed NOVX polypeptide is then recovered from the cells. Preferably, the cells produce little or no endogenous NOVX polypeptide. The cell can be, for example, a prokaryotic cell or a eukaryotic cell.

The present invention also provides for the NOVX polypeptide or NOVX nucleic acid in the sample by contacting the sample with a compound that specifically binds to the polypeptide or nucleic acid and detecting complex formation, if present. Relates to a method of identifying.

The invention further comprises contacting a NOVX polypeptide with a compound, and NOVX
By determining whether the activity of a polypeptide is modified, a method of identifying compounds that modulate the activity of a NOVX polypeptide is provided.

The invention also provides contacting a NOVX polypeptide with a compound, and the compound modifying the activity of the NOVX polypeptide, binding to the NOVX polypeptide, or a nucleic acid molecule encoding the NOVX polypeptide. To compounds that modulate NOVX polypeptide activity identified by determining if it binds to.

In another aspect, the invention provides a method of determining the presence of or predisposition to a NOVX-related disorder in a subject. The method includes providing a sample from a subject and measuring the amount of NOVX polypeptide in the subject sample. The amount of NOVX polypeptide in the subject sample is then compared to the amount of NOVX polypeptide in the control sample. A change in the amount of NOVX polypeptide in the subject's protein sample compared to the amount of NOVX polypeptide in the control protein sample indicates that the subject has a tissue growth associated condition. Control samples are preferably taken from comparable individuals (ie, individuals who are similar in age, sex, or other general condition, but who are not suspected of having a tissue growth-related condition). Alternatively, a control sample can be taken from a subject when the subject is not suspected of having a tissue growth-related disorder. In some embodiments, the NOVX is a NOVX.
Detected using an antibody.

In a further aspect, the invention provides a method of determining the presence of or predisposition to a NOVX-related disorder in a subject. This method is used for nucleic acid samples (eg,
RNA or DNA or both) from the subject, and measuring the amount of NOVX nucleic acid in the subject's nucleic acid sample. The amount of NOVX nucleic acid sample in the subject nucleic acid is then compared to the amount of NOVX nucleic acid in the control sample. A change in the amount of NOVX nucleic acid in the sample compared to the amount of NOVX in the control sample indicates that this subject has a NOVX-related disorder.

In yet a further aspect, the present invention provides methods of treating, preventing or delaying NOVX-related disorders. The method provides the subject, for whom such treatment or prevention or delay is desired, with a NOVX nucleic acid, NOVX polypeptide, or NOVX antibody sufficient to treat, prevent, or delay a NOVX-related disorder in the subject. Administration in an amount.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. . Although methods or materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel nucleotides and polypeptides encoded thereby. Novel nucleic acid sequences and their polypeptides are included in the present invention.
The sequences are collectively referred to as "NOVX nucleic acids" or "NOVX polynucleotides" and the corresponding encoded polypeptides are referred to as "NOVX polypeptides" or "NOVX proteins". Unless otherwise indicated, "NO
"VX" is meant to refer to any of the novel sequences disclosed herein. Table 1 provides a list of NOVX nucleic acids and the polypeptides they encode. Example 1 provides a description of how novel nucleic acids were identified.

[0021]

[Table 1] NOVX nucleic acids and the polypeptides they encode are useful in a variety of applications and contexts. Various NOVX nucleic acids and NOVX polypeptides according to the present invention are useful as novel members of the protein family according to the existence of domain and sequence relatedness to the proteins described above. In addition, NOVX nucleic acids and NOVX polypeptides can also be used to identify proteins that are members of the family to which NOVX polypeptides belong.

For example, VOVX1-3 are homologous to members of the collagen family of proteins that are important in determining cell shape and migration. Therefore, NOV1-3 nucleic acids and NOV1-3 polypeptides according to the invention, NOV1
The 3 antibodies and related compounds are useful in therapeutic and diagnostic applications in disorders characterized by altered cell motility, cell proliferation and cell migration (eg, cancer, angiogenesis and wound healing). .

NOV4 is also homologous to members of the potassium channel family of proteins present in all eukaryotic cells that maintain membrane potential and regulate electrical excitability in neurons. Accordingly, NOV4 nucleic acids and NOV4 polypeptides, NOV4 antibodies, and related compounds according to the invention may be used in neurological disorders such as episodic ataxia, autosomal dominant myokymia, stroke,
It is useful in therapeutic as well as diagnostic applications in Parkinson's disease, and Alzheimer's disease.

In addition, NOVX5-7 are enamel mineralizers
Tuftelin, a protein important for the alignment
It is homologous to family members. Therefore, NOV5 according to the present invention
7 nucleic acids and NOV5-7 polypeptides, NOV5-7 antibodies and related compounds are associated with disorders characterized by enamel deficiency (eg, enamel hypoplasia) and other disorders related to enamel deficiency (developmental and defective minerals). Chemical action (
(including hypominalization)) as well as therapeutic applications as well as diagnostic applications.

Furthermore, NOV8 is homologous to a family of neuronal antigen-like proteins that are important in tumor-associated neurological disorders. Therefore, N according to the invention
OV8 Nucleic Acids and NOV8 Polypeptides, NOV8 Antibodies and Related Compounds Treat in Disorders Characterized by Paraneoplastic Neurological Disorders (eg, Paraneoplastic Brainstem Limbic Encephalitis That Occurs During Testicular Cancer) It is useful in diagnostic as well as diagnostic applications.

NOV9-10 are also homologous to a family of fatty acid binding proteins that are important for keratinocyte differentiation. Therefore, NOVs 9-10 according to the invention
Nucleic acids and NOV9-10 polypeptides, NOV9-10 antibodies and related compounds find use in therapeutic and diagnostic applications in disorders characterized by aberrant keratinocyte differentiation, such as traumatic psoriatic skin. It is useful.

Finally, NOV11 is homologous to a family of cystatin-like proteins that are important in protecting eukaryotic cells from inappropriate proteolysis. Accordingly, NOV11 nucleic acids and NOV11 polypeptides, NOV11 antibodies and related compounds according to the present invention find use in therapeutic and diagnostic applications in disorders characterized by inappropriate proteolysis, such as atherosclerosis and abdominal aortic aneurysms. It is useful.

NOVX nucleic acids and NOVX polypeptides can also be used to screen for molecules that inhibit or enhance NOVX activity or function. In particular, the nucleic acids and polypeptides according to the invention are used for the identification of small molecules that regulate or inhibit, for example, neurogenesis, cell differentiation, cell motility, cell proliferation, hematopoiesis, wound healing and angiogenesis. Can be used as a target for

Additional utilities for NOVX nucleic acids and NOVX polypeptides according to the present invention are disclosed herein.

(VOV1) The NOV1 sequence according to the present invention is a nucleic acid sequence encoding a polypeptide related to the collagen family of proteins. NOV1 nucleic acids are expressed in the pancreas, salivary glands, lungs and lung tumors. The NOV1 nucleic acid and the polypeptide encoded thereby include the sequences shown in Table 2. The disclosed nucleic acid (SEQ ID NO: 1) is 1
, 949 nucleotides long and open reading frame (ORF
) (Starting at the ATG start codon at nucleotides 750 to 752 and ending at the TAG stop codon at nucleotides 1644-1646). A representative OPF encodes a 298 amino acid polypeptide (SEQ ID NO: 2) with a predicted molecular weight of 30,657.2 Daltons (Da). PSORT analysis of NOV1 polypeptides predicts cytoplasmic proteins with a certainty of 0.4500. The N-terminal signal sequence is obviously absent. The putative untranslated region upstream and downstream of the coding sequence is underlined in SEQ ID NO: 1.

[0031]

[Table 2] NOV1 polypeptide is human collagen type Iα (EMBL accession number: CAA
67261) and homology (33% identity), and Strongylocen
trotus purpuratus α-1 collagen (EM
It has homology (31% identity) with BL registration number: Q26634). The region of the NOV1 polypeptide also contains the human polypeptide sequence ORF6 as shown in Table 38.
52 (OPFX; PAtP registration number: B40888) and a high degree of homology (100%
Same).

[0032]

[Table 3] (NOV2) The NOV2 sequence according to the present invention is a nucleic acid sequence encoding a polypeptide related to the collagen family of proteins. NOV2 nucleic acids are expressed in the pancreas, salivary glands, lungs and lung tumors. NOV2 nucleic acids and the polypeptides encoded thereby include the sequences shown in Table 3. The disclosed nucleic acid (SEQ ID NO: 3) is 2
, 092 nucleotides long and open reading frame (ORF
) (Starting at the ATG start codon at nucleotides 767-769 and ending at the TAG stop codon at nucleotides 1616-1618). A representative OPF encodes a 283 amino acid polypeptide (SEQ ID NO: 4) with a predicted molecular weight of 29,009.5 daltons (Da). PSORT analysis of NOV2 polypeptides predicts cytoplasmic proteins with a certainty of 0.4500. The N-terminal signal sequence is obviously absent. The putative untranslated region upstream and downstream of the coding sequence is underlined in SEQ ID NO: 3.

[0033]

[Table 4] NOV2 polypeptide is human collagen type Iα (EMBL accession number: CAA
67261) and homology (33% identity), and Strongylocen
trotus purpuratus α-1 collagen (EM
It has homology (31% identity) with BL registration number: Q26634). The region of the NOV2 polypeptide also includes the human polypeptide sequence ORF652 (OPFX; PAt
It has a high degree of homology (100% identity) with P registration number: B40888).

(NOV3) A NOV3 sequence according to the present invention is a nucleic acid sequence encoding a polypeptide related to the collagen family of proteins. NOV3 was identified as described in Example 2 and is present in at least lymphoid tissue, mammalian gland / breast tissue, pancreas and salivary glands. NOV3 nucleic acids and polypeptides encoded thereby include the sequences shown in Table 4. The disclosed nucleic acid (SEQ ID NO: 5) is 1,011.
Nucleotides long and open reading frame (ORF) (starting at the ATG start codon at nucleotides 31-33 and at nucleotides 925-9
End with 27 TAG stop codons). A typical OPF is 29,009.
Encodes a 298 amino acid polypeptide (SEQ ID NO: 6) with a predicted molecular weight of 5 Daltons (Da). PSORT analysis of NOV2 polypeptide yields 0.4500
Predict cytoplasmic proteins with certainty. The N-terminal signal sequence is obviously absent. The putative untranslated region upstream and downstream of the coding sequence is underlined in SEQ ID NO: 5.

[0035]

[Table 5] One or more consensus positions (Conc. Pos.) Of the NOV3 nucleotide sequence are
It has been identified as a SNP as shown in Table 5. “Depth” indicates the number of clones covering the region of SNP. Putative Allele Frequency (Putative Alle)
le Freq. ) Is a fraction of all clones containing SNP. Symbol ">"
Means "is changed to".

[0036]

[Table 6] NOV3 polypeptide is human collagen type Iα (EMBL accession number: CAA
67261) with homology (33% identity), and as shown in Table 6, St
longylocentrotus purpuratus α
-1 collagen (COLa1; EMBL registration number: Q26634) and homology (3
1% identical, 39% similar). The region of the NOV3 polypeptide also shares a high degree of homology (99% identity) with the human polypeptide sequence ORF652 (OPFX; PAtP accession number: B40888).

[0037]

[Table 7] NOV1-3 are new members of the collagen family of proteins. NOV1-3 have a high degree of homology between each other as shown in Table 7 and thus represent a new subfamily of the collagen family of proteins. Collagen is the major structural glycoprotein of connective tissue. Unique primary structure and various post-translational modification reactions are required for normal fibril development. As post-translational modification,
Hydroxylation, glycosylation of prolyl and lysyl residues, molecular folding into a triple helix structure, proteolytic conversion of precursor procollagen to collagen, and oxidation of specific lysyl and hydroxylysyl residues. Deamination reaction is included. Any deficiency in the normal mechanisms leading to the synthesis and secretion of collagen molecules or the deposition of these molecules on extracellular fibers could result in abnormal fibril development; such defects could result in connective tissue disease. . Recently, deficiencies in enzymes involved in the regulation of types of synthesized collagen and post-translational modifications have been found in connective tissue inherited disorders. To some extent, the main inherited disorders of collagen metabolism in humans are lysyl hydroxylase deficiency in Ehrels-Dunlo syndrome type VI, p-collagen peptide deficiency in Ehrels-Dunlo syndrome type VII, type III collagen in Ehrels-Dunlo syndrome type IV. Decreased synthesis, lysyl oxidase deficiency in S-linked flaccid skin and Ehlers-Dunlo syndrome type V, and decreased type I collagen synthesis in osteogenesis imperfecta (PMID: 1448, UI.
: 76096101).

Unique collagen subtypes are recognized by specific cell surface receptors. Two of the most known collagen receptors are members of the integrin family and are called α1β1 and α2β1. The integrin α1β1 is abundant on smooth muscle cells, while the α2β1 integrin is the major collagen receptor on epithelial cells and platelets. Many cell types, such as fibroblasts, osteoblasts, chondrocytes, endothelial cells, and lymphocytes, can be concomitantly expressed with both of this receptor. Furthermore, the two receptors are connected to unique signaling pathways, and their ligation can lead to opposite cellular responses (see, for example, PMID: 10963992).

Connective tissue maintains its shape against external and internal stress. These are molecular graded organizations in which the basic building blocks are grouped into layers that increase in complexity and interaction based on information carried by precursor molecules secreted by cells.
Collagen fibrils are well-understood self-aggregation targets controlled by their amino acid sequences, while interfibrillar amorphous substrates are the primary structure of the characteristic glycosaminoglycans dissolved therein. It has not been previously seen as composed of similar aggregates defined by Transmission electron microscopy using morphometry and stereoanalysis has demonstrated their presence in tissues. Nuclear magnetic resonance defines these secondary structures, rotating shadowing electron microscopy describes their aggregation in vitro, and molecular dynamics stimuli show how the latter can arise from the former. Indicated. The driving forces for aggregation are offset by hydrophobic and hydrophilic bonds, the electrostatic reciprocity between the charges of the polyanions. The relative stability of aggregation is determined by this balance and therefore by the position and number of their charges (especially the sulfate ester groups). The corneal stroma is a system of collagen fibrils and is highly aligned to ensure transparency, where glycosaminoglycan aggregation is organized as a reference, in a manner that has similarities in all connective tissues. Have been proposed (PMID: 1612287, UI: 9230).
7242).

Recent biochemical and immunohistochemical studies have shown that heparan sulfate proteoglycans, two high molecular weight glycoproteins (fibronectin and laminin), and 2
Several components of the basement membrane are described, including two collagen types (type IV and V). These collagens have several properties that distinguish them from other types located in the stroma: (a) Type IV forms an amorphous felt-like matrix, and either IV or V , Not found in fibrils with crossed bands, (b) both have increased content of hydrophobic amino acids, (c) precursor (pro) form is larger than interstitial collagen, (d) ) Type IV has a break in the triple helix, and (e) both IV and V are resistant to human skin collagenase, but selected neutral from mast cells, macrophages, and granulocytes. It is a substrate for proteases. By immunofluorescence staining, IV collagen was localized under the basement membrane at the dermal-epithelial junction of the capillaries and endothelial cells in the larger blood vessels. Ultrastructurally, it was shown to be a specific component of the basement membrane compact layer. Collagen V is localized to the pericellular matrix of some cell types and may be specific for the extramembrane structure closely associated with the basement membrane. Other collagen proteins that may be associated with the extracellular matrix have been described. One of these is secreted by endothelial cells in culture and exhibits a novel collagen type by peptide mapping. It is secreted under ascorbic acid-free conditions and is highly susceptible to proteolysis. It has been proposed that kinetic reciprocity exists between cells and their extracellular matrix, which partially determines cell shape, biosynthesis, migration, and adhesion. Examples of phenotypic regulation in some of these events have been shown using endothelial cells grown on different substrates and isolated from different vascular environments (eg PMID:
7045245, UI: 82215350).

NOV1-3 represent a new subfamily of the collagen family. NO
V1-2 can be used to detect pancreas, salivary glands, lungs and lung tumors, and NOV
3 can be used to detect at least lymphoid tissue, mammary gland / breast tissue, pancreas and salivary glands. NOV1-3 are useful in determining changes in expression of genes contained within the collagen protein family. NOV1-3 fills a need in the art by providing new diagnostic or therapeutic compositions that are useful in the treatment of disorders associated with alterations in the expression of members of collagen-related proteins. NOV1-3 nucleic acids, polypeptides, antibodies, and other compositions of the invention include, by way of non-limiting example, pancreatic cancer, breast cancer, lymphoma, and other disorders characterized by alterations in cell shape, motility and differentiation. It is useful in the treatment and / or diagnosis of various diseases and conditions, including (eg, pathological angiogenesis and wound healing).

[0042]

[Table 8] Where * indicates identity and: indicates strong similarity.

(NOV4) The NOV4 sequence according to the present invention is a nucleic acid sequence encoding a polypeptide related to a protein of the voltage-gated potassium channel-like protein family. NOV4
The nucleic acid is found on human chromosome 19. NOV4 nucleic acids and their encoded polypeptides include the sequences shown in Table 8. The disclosed nucleic acid (SEQ ID NO: 7) is 1,747 nucleotides long and has an A at nucleotides 38-40.
TGA starting at the TG start codon and at nucleotides 1715-1717
It contains an open reading frame (ORF) ending with a stop codon. A representative ORF encodes a 559 amino acid polypeptide (SEQ ID NO: 8) with a predicted molecular weight of 61,642.7 Da. PSORT analysis predicts that NOV4 polypeptide is a plasma membrane protein (certainty 0.6000). The putative untranslated region upstream and downstream of the coding sequence is underlined in SEQ ID NO: 7.

[0044]

[Table 9] NOV4 nucleic acid is cloned CTB-60B18, as shown in Table 9.
(CHR19; GenBank accession number: AC008687), including human first
It has a high degree of homology (100% identity) with the uncharacterized region of 9 chromosomes. The NOV4 polypeptide is a mouse-derived voltage-gated potassium channel-like protein (VGPC; EMBL accession number: AAC2), as shown in Table 10.
3664) with homology (83% identity, 85% similarity). NOV4 polypeptides also share homology (69% identity, 81% similarity) with voltage-gated potassium channel-like protein (HGK5; EMBL accession number: P22001), as shown in Table 11.

[0045]

[Table 10] Where * indicates identity and + indicates similarity.

[0046]

[Table 11]

[0047]

[Table 12] Where * indicates identity and + indicates similarity.

NOV4 represents a new member of the subclass of voltage-gated potassium channels, including members from several species (eg, human, mouse and rat), as shown by CLUSTAL W analysis in Table 12.

[0049]

[Table 13] Here, * indicates identity ,: indicates strong similarity, and-indicates weak similarity. Rat potassium channel KV1.3 (rKV; registration number: A4353
10); human potassium channel KV1.3 (hKV; registration number: P22001)
Mouse potassium channel KV1.7 (mKV; accession number: AAC23664)
.

Potassium channels represent the most complex class of voltage-gated ion channels, both from a functional and structural perspective. Present in all eukaryotic cells, their diverse functions include maintaining membrane potential, regulating cell volume, and regulating electrical excitability of neurons. The delayed rectifying function of potassium channels allows neuronal cells to efficiently repolarize after an action potential. Drosop
In hila, four sequence-related K + channel genes (Shaker, Sha
w, Shab, and Shal) have been identified. Each gene has been shown to have a human homolog.

By PCR of genomic DNA using primers based on a conserved region between the Drosophila Shaker and the mouse voltage-gated potassium channel,
Ramaswami and collaborators (Ramaswami et al., 1990, Mo.
l. Cell. Nuorsci. 1: 214) isolated fragments of several related human genes. They used the fragments to screen a cDNA library, and they used HuKI (KCNA1), H
uKII (KCNA4; 176266), HuKIV (KCNA2; 17626)
2), and a cDNA encoding several potassium channels designated HuKV (KCNA6; 176257). Like other Shaker-class potassium channels, the putative 495 amino acid KCNA1 protein contains 6
Hydrophobic segment, S4 between hydrophobic segment 3 and hydrophobic segment 4
Contains the positively charged region, called, and the leucine zipper. KCNA1 shares 98% amino acid identity with its rat homolog RCK1. When expressed in Xenopus oocytes, KCNA1, KCNA4, and KCNA2
It showed different voltage dependence, kinetics, and sensitivity to pharmacological potassium channel blockers. KCNA1 and KCNA2 are non-inactivated channels, resembling delayed rectification, but KCNA4 was inactivated rapidly.

Chandy and co-workers (Chandy et al., 1990, Science).
247: 973) are three closely related potassium channel genes (MK1).
, MK2, and MK3) were located at distinct sites in the mouse genome. These genes, which encode the subunits of the voltage-gated K + channel, are homologous to the Drosophila Shaker gene. Curra
(Curran et al., 1992, Genomics 12: 729) have mapped the KCNA1 gene to chromosome 12 by using a human-rodent somatic cell panel and mapped it to the distal short arm by in situ hybridization. Narrowed. Linkage studies include KCNA1 and von Willeb
1. at a recombination ratio of 0.05 with the rand locus (VWF; 193400).
A maximum rod score of 72 was shown. Using interspecific backcrosses between Mus musculus, Klocke et al. (Klocke et al., 1993, Genomics).
18: 568) describes the Kcna1, Kcna5 (176267), and Kcna6 genes on mouse chromosome 6 near the homologue of TPI1 (190450), which is located on human chromosome 12 p13. Mapped. Albrecht and coworkers (Albrecht et al., 1995, Rec
eptors Channels 3: 213), chromosome 12p.
Human KCNA6, KCNA with 300 kb clusters on 13 arranged in tandem
1 and KCNA5 gene.

Browne et al. (Browne et al., 1994, Nature Genetics).
s 8: 136) is myokymia (muscle rippling) ()
Mutational analyzes of the KCNA1 coding region of four families with type 1 accidental ataxia (also known as EA1; 160120) were also performed. They found four different missense mutations that exist in a heterozygous state. For a comprehensive review of incidental ataxia type 1 and its underlying mutations, see Bran.
dt and Strupp, 1997, Audiol. Neuroroot. 2: 3
See 73. Adelman et al. (Adelman et al. 1995, Neur
on 15: 1449), cDNAs corresponding to six different mutations associated with autosomal dominant myokymia with accidental ataxia were added to Xenopus ocy.
injected into tes. They demonstrated that co-assembly of one or more accidental ataxia subunits with wild-type subunits could alter channel machinery and confer a dominant negative effect.

NOVX4 is a new member of the voltage-gated potassium channel-like protein family of proteins. It is useful as a marker for human chromosome 19. As a member of the voltage-gated potassium channel-like family of proteins, N
OV4 nucleic acids, proteins, antibodies and other compositions of the present invention can be used to detect accidental ataxia type 1, long QT syndrome 1 and long QT syndrome 2, benign neonatal epilepsy, Gerber-Langernielsen syndrome, autosomal dominant hearing loss ( DFNA2), non-insulin dependent diabetes mellitus, CNS disorders, arrhythmias, stroke, asthma, hypertension treatment and / or potential therapeutic applications involving other conditions and disorders. NOV
4 may be useful in drug screening for the identification of therapeutics that modulate channels and thus regulate insulin secretion. Selective antagonists increase insulin release, thereby reducing hyperglycemia associated with non-insulin dependent diabetes.

(NOV5) A NOV5 sequence according to the present invention is a nucleic acid sequence encoding a polypeptide related to a protein of the tufterin-like protein family. NOV5 nucleic acids are expressed in fetal liver, testis, fetal lung, and thyroid. NOV5 nucleic acids and their encoded polypeptides include the sequences shown in Table 13. The disclosed nucleic acid (SEQ ID NO: 9) is 1,080 nucleotides long and contains nucleotides 45
It contains an open reading frame (ORF) that begins at the ATG start codon at -47 and ends at the TGA stop codon at nucleotides 799-801. A representative ORF encodes a 251 amino acid polypeptide (SEQ ID NO: 10) with a predicted molecular weight of 29,229.5 Da. PSORT analysis of NOV5 predicts a cytoplasmic protein (certainty 0.4500), and NOV5 appears to lack the N-terminal signal sequence. The putative untranslated region upstream and downstream of the coding sequence is underlined in SEQ ID NO: 9.

[0056]

[Table 14] The NOV5 nucleic acid is human cutaneous T cell lymphoma associated antigen se57-1 mRNA (CTCL; GenBank Accession No: AF27) as shown in Table 14.
3051) and a high degree of homology (99% identity). The NOV5 polypeptide has homology (26% identity, 55% similarity) with the boss taurus chufterin-like protein (bTUF; EMBL accession number: O97683), as shown in Table 15.

[0057]

[Table 15] Where * indicates identity and + indicates similarity.

(NOV6) The NOV6 sequence according to the present invention is a nucleic acid sequence encoding a polypeptide related to a protein of the tufterin-like protein family. NOV6 nucleic acids are expressed in fetal liver, testis, fetal lung, and thyroid. NOV6 nucleic acids and their encoded polypeptides include the sequences shown in Table 16. The disclosed nucleic acid (SEQ ID NO: 11) is 1,482 nucleotides long. The opposite complement of SEQ ID NO: 11 is SEQ ID NO: 69. The NOV6 nucleic acid begins at the ATG start codon at nucleotides 195-197 of SEQ ID NO: 69 and at nucleotide 1
It contains an open reading frame (ORF) ending with the TGA stop codon at 199-1201. A representative ORF encodes a 335 amino acid polypeptide (SEQ ID NO: 12) with a predicted molecular weight of 38,839.1 Da. NOV6
PSORT analysis predicts a cytoplasmic protein (certainty 0.4500), and NOV6 appears to lack the N-terminal signal sequence. The putative untranslated region upstream and downstream of the coding sequence is underlined in SEQ ID NO: 69.

[0059]   (Table 16)

[0060]

[Table 16] The NOV6 nucleic acid is a human cutaneous T cell lymphoma associated antigen se57-1 mRNA (
CTCL; GenBank accession number: AF273051) and a high degree of homology (99
% Identity). The NOV6 polypeptide has homology (25% identity, 53% similarity) with bos taurus chufterin-like protein (bTUF; EMBL accession number: O97683).

(NOV7) A NOV7 sequence according to the present invention is a nucleic acid sequence encoding a polypeptide related to a protein of the tufterin-like protein family. NOV7 nucleic acids are expressed in fetal liver, fetal lung, testis, fetal lung, and thyroid. NO
V7 nucleic acids and their encoded polypeptides include the sequences shown in Table 17. The disclosed nucleic acid (SEQ ID NO: 13) is 1,442 nucleotides long and contains an open reading frame (ORF) that begins at the ATG start codon at nucleotides 155-157 and ends at the TGA stop codon at nucleotides 1159-1161. A representative ORF encodes a 335 amino acid polypeptide (SEQ ID NO: 14) with a predicted molecular weight of 42,276.8 Da. NOV
PSORT analysis of 7 predicts a cytoplasmic protein (certainty 0.4500), and NOV7 appears to lack the N-terminal signal sequence. The putative untranslated region upstream and downstream of the coding sequence is underlined in SEQ ID NO: 13.

[0062]   (Table 17)

[0063]

[Table 17] The NOV7 nucleic acid is a human cutaneous T cell lymphoma associated antigen se57-1 mRNA (
CTCL; GenBank accession number: AF273051) and a high degree of homology (99
% Identity). The NOV7 polypeptide has homology (26% identity, 55% similarity) with bos taurus chufterin-like protein (bTUF; EMBL accession number: O97683).

The NOV5-7 polypeptides have a high degree of homology between each other, as shown in Table 18. Therefore, NOVs 5-7 represent a novel subfamily of the tufterin-like protein family.

[0065]   (Table 18)

[0066]

[Table 18] Where * indicates identity ,. Indicates weak similarity.

NOV5-7 have similarities to the enamelin family of proteins, chufterin. Chufterin is a novel acidic enamel protein believed to play a major role in enamel mineralization and is involved in the pathogenesis of autosomal inherited enamel hypoplasia (AI). AIs are a diverse group of genetic disorders characterized by various enamel defects, including underdevelopment and defective mineralization.

Chufterin is a novel acidic enamel protein believed to play a major role in enamel mineralization. Its identification and location is determined by amino acid composition, enzyme-linked immunosorbent assay, Western blot, indirect immunohistochemistry (indirec).
immunohistochemistry, and high resolution protein A gold immunocytochemistry (high resolution protein N-Ag).
old immunocytochemistry). The putative tufterin protein (pI5.2) contains 389 amino acids, and 43,
It has a calculated peptide molecular weight of 814 Da. Immunological studies indicate conservation of chufterin structure between species throughout vertebrate evolution. This cDNA sequence localizes to various putative post-translational sites (including one N-glycosylation consensus site, seven O-glycosylation sites, and seven phosphorylation sites) and N-terminal region. It encodes the hand calcium binding domain (mismatch). In the C-terminal region (residues 252-345), chufterin contains structurally apparent determinants for self-assembly. The human tufuterin gene was mapped to chromosome 1 q21-31 using fluorescence in situ hybridization. Localization of the human tufuterin gene to a well-defined cytogenetic region is important for understanding the etiology of autosomal inherited enamel hypoplasia, the most common enamel inherited disease (Deutsch et al., 1997, Ciba
Found Symp 205: 135-47; Discussion 147-1.
55).

The bovine tufterin gene was compared to that of the bovine tufterin cDNA.
This analysis showed that the cDNA had a calculated molecular weight of 38,630 and a 5.85.
It was demonstrated to contain a 1014 bp open reading frame encoding a 338 residue protein with an isoelectric point of. These results differ from previously published results that included different conceptual amino acid sequences for the carboxy-terminal region and identified different terminal codons. Prior to the present invention, the bovine tufterin protein was not believed to share homology or domain motifs with any other known protein. This gene consists of 13 exons ranging in size from 66 to 1531 bp, the latter containing the encoded carboxy-terminal region and the 3'untranslated region. Exons are integrated more than 28 kbp of genomic DNA. Codons are generally not split at exon / intron boundaries.
Various alternative spliced transcripts were identified by DNA sequence analysis of the isolated products produced by the reverse transcriptase / polymerase chain reaction (Bashi
r et al., 1997, Arch Oral Biol 42: 489).
.

NOV5-7 are novel members of the tufterin-like family of proteins. NOVs 5-7 are useful in detecting fetal liver, testis, fetal lung, and thyroid tissue. The expression patterns of NOV5-7 and other members of the chufterin-like protein family, and the similarity of the genes to the enamelin protein family, indicate that NOV5-7 can function as enamel proteins in expressing tissues. It is therefore considered relevant in disorders involving these tissues (eg, enamel hypoplasia) and other disorders associated with enamel deficiency (including underdevelopment and hypomineralization).

(NOV8) The NOV8 sequence according to the present invention is a nucleic acid sequence encoding a polypeptide related to a protein of the neuronal antigen-like protein family. The NOV8 nucleic acid was mapped to human chromosome 14. NOV8 nucleic acids are expressed at least in the brain, brain stem and testis. NOV8 nucleic acids and their encoded polypeptides include the sequences shown in Table 19. The disclosed nucleic acid (SEQ ID NO: 15) is 1,05
It is 6 nucleotides long and contains an open reading frame (ORF) that begins at the ATG start codon at nucleotides 1-3 and ends at the TGA stop codon at nucleotides 1,054-1,056. Typical ORF is 351
Encodes the amino acid polypeptide (SEQ ID NO: 16). PSORT analysis is NO
It is presumed that the V8 polypeptide is localized in the mitochondrial membrane space (certainty 0.3600).

[0072]   (Table 19)

[0073]

[Table 19] NOV8 nucleic acid is cloned into RPCI4-794B2 as shown in Table 20.
Uncharacterized region of human chromosome 14 (CHR14; GenBa
It has a high degree of homology (100% identity) with nk accession number: AC005924). The NOV8 polypeptide is homologous (5) to human tumor-associated neuronal antigen protein (hPNA; EMBL accession number: O95144), as shown in Table 21.
5% identity, 72% similarity). Also, as shown in Table 22, the NOV8 polypeptide is a human tumor-associated neuron antigen mm2 polypeptide (hP
NA; EMBL accession number: O95145) with homology (49% identity, 65% similarity). In addition, regions of the NOV8 polypeptide also have a high degree of homology (100% identity) with the human polypeptide sequence ORF2787 (ORFX; PatP accession number B43023), as shown in Table 39.

[0074]   (Table 20)

[0075]

[Table 20] (Table 21)

[0076]

[Table 21] Here, * indicates identity and + indicates similarity.

[0077]   (Table 22)

[0078]

[Table 22] Here, * indicates identity and + indicates similarity.

[0079]   (Table 39)

[0080]

[Table 23] Here, * indicates identity.

In patients with cancer, symptoms of limbic or brainstem dysfunction can result from paraneoplastic disorders. Paraneoplastic limbic and brainstem encephalitis occurs more often with testicular cancer than most other cancers. Anti-neuronal antibodies can be used in diagnostic tests for this syndrome. Using immunohistological and immunoblotting techniques,
Serum antibodies and cerebrospinal fluid antibodies were detected. Serological screening of complementary DNA libraries and Northern blots were used to clone the target antigen and to determine which tissues express it. Of the 13 patients with testicular cancer and paraneoplastic limbic or brainstem (or both) encephalitis, 10 had antibodies to the 40-kd neuronal protein in serum and cerebrospinal fluid. These antibodies were used to clone a gene called Ma2. This gene encodes a protein (Ma2) that was recognized by sera from 10 patients but not by sera from 344 control subjects. M
a2 is selectively expressed by normal brain tissue and patient testicular tumors. Ma
2 shares homology with Ma1, a "brain-testis-cancer" gene associated with other paraneoplastic syndromes and tumors. Thus, the sera of patients with subacute limbic and brainstem dysfunction and testicular cancer contain antibodies to the proteins found in normal brain and testicular tumors. Detection of these antibodies supports the paraneoplastic origin of neurological disorders and may have diagnostic significance (Voltz et al., 1
999, N.M. Engl. J. Med. 340: 1788).

Identification of anti-neuronal antibodies also facilitated diagnosis of paraneoplastic neurological disorders, and early detection of associated tumors. It also led to the cloning of potentially important neuron-specific proteins. Serological studies of 1705 sera from patients with expected paraneoplastic disorders show 37 kDa and 40 kDa.
Four patients were identified with antibodies that reacted with the neuronal protein of a (anti-Ma antibody). Three patients had brainstem and cerebellar dysfunction, and one had dysphagia and motor weakness. Autopsy dissection of two patients, Purkinje cells,
Bergmann showed gliosis and loss of deep cerebellar white matter inflammatory infiltrates. Mainly CD8
Extensive neurodegeneration, gliosis, and invasion composed of + T cells is also 1
Found in the brainstem of a human patient. In normal human and rat tissues, anti-Ma antibodies react exclusively with neurons and testicular germ cells; this reaction is primarily associated with nuclear elements (including nuclear inclusions) and to a lesser extent. With cytoplasm. Anti-Ma antibodies also reacted with cancers (breast, colon, and parotid gland) available from 3 anti-Ma patients, but not with 66 other tumors of altered histological type. . Pre-incubation of tissues with any anti-Ma serum eliminated the reactivity of other anti-Ma immunoglobulins. Probing a human complementary DNA library with anti-Ma serum resulted in the cloning of the gene encoding the novel 37 kDa protein (Ma1). Recombinant Ma1 was specifically recognized by 4 anti-Ma sera, but 337 control sera (52 normal individuals, 179 cancer patients without paraneoplastic neurologic symptoms, paraneoplastic) With symptoms of 9
Includes sera from 6 patients and 10 patients with non-cancer related neuropathy)
Was not recognized by. Expression of Ma1 mRNA is highly restricted to the brain and testis. Subsequent analysis indicated that Ma1 may be a phosphoprotein. Some patients with paraneoplastic neurological disorders develop antibodies to Ma1, a novel member of the expanded family of "brain / testis" proteins.

NOV8 is a novel member of the neuronal antigen-like protein family of proteins. The expression pattern of the NOV8 gene and its family members,
And its similarity to the neuronal antigen-like protein family of genes,
It is shown that NOV8 can function as a neuronal antigen in expressing tissues. NO
V8 is useful as a marker for brain tissue, brainstem tissue, and testis tissue. Therefore, NOV8 is implicated in disorders involving these tissues. Some of these disorders include, but are not limited to: cardiovascular disorders, diabetes, leukemia / lymphoma, cancer, musculoskeletal disorders, muscle degeneration, reproductive health, metabolic and endocrine disorders, gastrointestinal disorders, immunity. Disorders and autoimmune diseases, respiratory disorders, bone disorders,
And disorders of tissue / cell growth regulation. NOV8 is also useful as a marker for human chromosome 14.

(NOV9) The NOV9 sequence according to the present invention is a nucleic acid sequence encoding a polypeptide related to a protein of the fatty acid binding protein-like protein family. NOV9 nucleic acids and their encoded polypeptides include the sequences shown in Table 23. The disclosed nucleic acid (SEQ ID NO: 17) is 499 nucleotides long and begins with the ATG start codon at nucleotides 5-7 and TAA at nucleotides 494-496.
It contains an open reading frame (ORF) that ends with a stop codon. A representative ORF encodes a 163 amino acid polypeptide (SEQ ID NO: 18). The coding sequence upstream of the putative untranslated region and the coding sequence downstream of the putative untranslated region are underlined in SEQ ID NO: 17. PSORT analysis predicts that the NOV9 polypeptide localizes to the cytoplasm (certainty 0.6500). SIGNALP analysis suggests that the NOV9 polypeptide has no signal peptide.

[0085]   (Table 23)

[0086]

[Table 24] The NOV9 nucleic acid has a high degree of homology (92% identity) with the human fatty acid binding protein homolog mRNA (hFBP; GenBank accession number M94856), as shown in Table 24. NOV9 nucleic acids are also shown in Table 25 as shown in Table 25.
Human melanogenesis inhibitor mRNA (hMI; PatP accession number R5586
It has a high degree of homology with 6) (94% identity). The NOV9 polypeptide is a human epidermal fatty acid binding protein (eFBP; Swiss), as shown in Table 26.
It has homology (88% identity, 92% similarity) with Prot accession number Q01469).

[0087]   (Table 24)

[0088]

[Table 25] (Table 25)

[0089]

[Table 26] (Table 26)

[0090]

[Table 27] Here, * indicates identity and + indicates similarity.

(NOV10) The NOV10 sequence according to the present invention is a nucleic acid sequence encoding a polypeptide related to a protein of the fatty acid binding protein-like protein family. NOV1
0 nucleic acids and their encoded polypeptides include the sequences shown in Table 27.
The disclosed nucleic acid (SEQ ID NO: 19) is 413 nucleotides long and begins with an ATG start codon at nucleotides 6-8 and a T at nucleotides 408-410.
It contains an open reading frame (ORF) ending at the AA stop codon. A representative ORF encodes a 134 amino acid polypeptide (SEQ ID NO: 20).
PSORT analysis showed that the NOV10 polypeptide was localized in the cytoplasm (0.6
(A certainty of 500) and SIGNALP analysis suggests that the NOV10 polypeptide has no signal peptide. The coding sequence upstream of the putative untranslated region and the coding sequence downstream of the putative untranslated region are underlined in SEQ ID NO: 19.

[0092]

[Table 28] NOV10 nucleic acid is a human fatty acid binding protein homolog mRNA (GenB
It has a high degree of homology (94% identity) with ank accession number M94856). N
The OV10 nucleic acid also has a high degree of homology (94% identity) with the human melanogenesis inhibitor mRNA (PatP accession number R55866). The NOV10 polypeptide can bind to human epidermal fatty acid binding protein (eF) as shown in Table 28.
BP; homology with SwissProt accession number Q01469) (88% identity, 9
2% similarity).

[0093]

[Table 29] NOV9-10 are highly homologous to each other and CL in Table 29
It is highly homologous to other members of the fatty acid binding protein-like family of proteins as shown by USTALW analysis.

[0094]

[Table 30] Where * indicates identity ,: indicates strong similarity, and. Indicates weak homology. Rat fatty acid binding protein (rFABP; SwissProt registration number P
55053), mouse fatty acid binding protein (mFABP; SwissProt
Registration number Q05816), and human fatty acid binding protein (hFABP; Sw
(IssProt registration number Q01469).

Fatty acid metabolism in mammalian cells depends on the flux of fatty acids between the plasma membrane and the mitochondria or peroxisomes for β-oxidation, as well as between other cellular organelles for lipid synthesis. . Fatty acid binding protein (FA
The BP) family is composed of small cytosolic proteins thought to be involved in uptake, transport, and solubilization of these hydrophobic ligands. Members of this family have highly conserved sequences and tertiary structure. Fatty acid binding proteins were first isolated in the intestine (FABP2; OMIM-134640) and later in the liver (FABP1; OMIM-134650), striated muscle (F.
ABP3; OMIM-134651), adipocytes (FABP4; OMIM-60)
0434) and epidermal tissue (E-FABP; GDB ID: 136450).

Epidermal fatty acid binding protein (E-FABP) was cloned from the skin of patients with psoriasis, as was the novel keratinocyte protein by Madsen and coworkers (Madsen et al., 1992, J. Invest. Dermato).
l. 99: 299). Later, using quantitative Western blotting analysis, Kingma et al. Showed that in addition to skin, bovine E-FABP is expressed in retina, testis, and lens (Kngma et al., 1998, Bio.
Chemistry 37: 3250). Since E-FABP was originally identified from the skin of psoriasis patients, it is also known as psoriasis-related fatty acid binding protein (PA-FABP). PA-FABP is a cytoplasmic protein and is expressed on keratinocytes. It is highly upregulated in psoriatic skin. It shares similarities with other members of the fatty acid binding protein and belongs to the fabp / p2 / crbp / crabp family of transporters. PA-FABP has a high specificity for fatty acids and is believed to have the highest affinity for c18 chain length. Reducing chain length or introducing double bonds reduces affinity. PA-FABP may be associated with keratinocyte differentiation.

Immunohistochemical localization of E-FABP expression in psoriasis, basal cell carcinoma and squamous cell carcinoma has been performed to gain indirect information about fatty acid transport at the cellular level. (Masouye et al., 1996, Dermatology
192: 208). E-FABP was localized to the upper spinous and granular layers in normal and intact psoriatic skin. In contrast, damaged psoriatic epithelium
E-FABP was strongly expressed like non-keratinized oral mucosa in all suprabasal layers. The basal layer did not express E-FABP reactivity in any of these samples. Thus, although basal cell carcinoma was E-FABP negative, only well differentiated cells of squamous cell carcinoma expressed E-FABP. This indicates that E-FABP expression is associated with the restriction of keratinocyte differentiation, and E
Suggesting that the putative role of FABP should not be restricted to the formation of the skin lipid barrier. Since the pattern of E-FABP expression mimics cellular FA transport, injured psoriatic skin and oral mucosa may have higher metabolism / transport for FA than normal and undamaged psoriatic epithelium.

NOV9-10 represent a new member of the family of epidermal fatty acid binding proteins and are therefore useful for determining epidermal fatty acid binding proteins that interact with the protein. The expression patterns of the NOV9-10 genes and their family members and their similarities to genes of the epidermal fatty acid binding protein family indicate that they are involved in fatty acid synthesis, uptake, transport, localization in expressed tissues (eg keratinocytes), It is suggested that these hydrophobic ligands may function as regulators of solubilization. NOV9-10 are useful as markers for keratinocyte differentiation. NOVs 9-10 are thus implicated in diseases associated with these tissues. Some of these diseases include, but are not limited to, damaged psoriatic skin and damaged oral mucosa.

(NOV11) The NOV11 sequence according to the present invention is a nucleic acid sequence encoding a polypeptide related to the cysteine family of proteins. NOV11 nucleic acids and their encoded polypeptides include the sequences shown in Table 30. The disclosed nucleic acid (SEQ ID NO: 21) is 468 nucleotides long and has an A of nucleotides 10-12.
It contains an open reading frame (ORF) that begins at the TG start codon and ends at the TGA stop codon at nucleotides 445-447. Typical ORF is 1
It encodes a 45 amino acid polypeptide (SEQ ID NO: 22). The mature form of the NOV11 polypeptide is described in Example 3. The upstream and downstream of the putative untranslated region of the coding sequence are underlined in SEQ ID NO: 21. PSORT analysis is NOV1
Predict that 1 polypeptide is a secreted protein (certainty 0.60
42), SIGNALP analysis suggests that NOV11 has an N-terminal signal peptide with a cleavage site probably present between positions 19 and 20 of SEQ ID NO: 22.

[0100]

[Table 31] The NOV11 nucleic acid is labeled on human chromosome 20p11.21-1 as shown in Table 31.
It has a high degree of homology (100% identity) with the region of 2.3 (including clone RP3-322G13 (CHR20; GenBank accession number: HSJ322G13)). The NOV11 polypeptide has rat cysteine C as shown in Table 32.
It is homologous to the polypeptide (RCYS; GenBank Accession No. P14841). NOV11 also binds to human cystatin D polypeptide (as shown in Table 33).
hCYS; GenBank accession number P28325).

[0101]

[Table 32]

[0102]

[Table 33]

[0103]

[Table 34] The superfamily of cysteine proteinase inhibitors includes structurally homologous proteins that are divided into at least three families: family I (stefin; see OMIM 184600), family II (cystatin), and family. III (kininogen; OMIM 2
28960). Salivary cystatin (cystatin S, SA and SN
, Known mainly as saliva, tears and seminal plasma,
ST3; OMIM 604 312) is abundantly present in cerebrospinal fluid, seminal fluid, milk, synovial fluid, and urine as well as plasma in patients with uremia. These proteins may play important roles in protecting cells from inappropriate proteolysis and in regulating cysteine proteinases of both host and bacterial origin. Saitoh and coworkers (Saitoh et al., 1987, Gene.
61: 329) have shown that salivary form of cystatin is determined by a gene family consisting of at least 7 loci. They have three cystatin genes, CST1 for cystatin SN, CS for cystatin SA
CSTP1 for T2, and the cystatin pseudogene were isolated. Saito
h et al. that CST3, a gene encoding cystatin C and a variant in cerebral amyloid angiopathy of the Icelandic type (OMIM 105150), has the same mechanism as the CST1 and CST2 genes. (Saitoh et al., 1989, Biochem. Biophy.
s. Res. Commun. 162: 1324). Southern analysis of somatic hybrid clones demonstrated that all members of the cystatin gene family segregate on human chromosome 20.

Cystatin C, which belongs to the type II cystatin gene family, is the most abundant extracellular inhibitor of cystatin protease. It is a 13 kDa protein that is secreted constitutively shortly after its synthesis (Barrett et al., 19).
84, Biochem. Biophys. Res. Commun. 120: 63
1). Grubb and Lofberg reported the amino acid sequence of this protein (Grubb and Lofberg, 1982, Proc.
Nat. Acad. Sci. 79: 3024). The isolation and characterization of six human cysteine proteinase inhibitors, including cystatin C, was reported in 1988 (Abrahamson, 1988, Scand.
J. Clin. Lab. Invest. Sups. 191: 21)
. Cystatin D (OMIM 123858), S (OMIM 123857),
And SA (OMIM 123856) are expressed predominantly in the salivary glands, whereas Cystatin C is expressed in virtually all organs of the body. Due to its high concentration in biological fluids, cystatin C is probably the most important extracellular inhibitor of cysteine proteinases. Cystatin C is present in many neuroendocrine cells and its concentration in cerebrospinal fluid is 5.5 times that in plasma of healthy adults (Grubb and Lofberg, 1982, Proc. Na.
t. Acad. Sci. 79: 3024).

Atherosclerosis and Aortic Aneurysm (AAA; OMIM 100070)
Is associated with the destruction of elastic lamina. Elastic fibrinolytic cysteine protease (
Although cathepsins S and K) are overexpressed at the site of arterial elastin injury, it is unknown whether endogenous, local inhibitors balance these proteinases. Shi and colleagues show that cystatin C is normally expressed on smooth muscle cells of the vascular wall, but that this cysteine protease inhibitor is highly reduced in both atherosclerosis and aortic aneurysm injury. (Shi et al., 1999, J. Clin. Invest. 104: 1191). Moreover, the increase in abdominal aneurysm diameter of 122 patients screened by ultrasonography was inversely proportional to serum cystatin C levels. In vitro, cytokine-stimulated vascular smooth muscle cells secrete cathepsins and their elastolytic activity could be inhibited when cystatin C secretion was induced by treatment with TGF-β-1. These findings highlighted a potentially important role for the imbalance between cysteine proteases and cystatin C in the remodeling of the arterial wall, and established that cystatin C deficiency occurs in vascular disease. . Shi et al.
To i. et al., significant inhibition of cystatin C concomitant with the increased expression of cysteine proteases observed in their studies in atherosclerosis and abdominal aneurysm was due to the first acquired cysteine in human disease It has been reported to exhibit protease inhibitor deficiency (Shi et al., 1999, J. Cli.
n. Invest. 104: 1191).

NOV11 represents a novel member of the cystatin family of proteins. Thus, NOV11 is useful in identifying proteins that interact with cysteine. NOV11 is also useful as a marker for the region of human chromosome 20p11.21-11.3.

Nucleic acids and proteins of the invention are found to have abnormal cell proliferation, cell differentiation and migration (eg, cancer, angiogenesis and wound healing), neurological disorders (eg, tumor-associated neurological injury, Apocalyptic ataxia, autosomal dominant myokimia, stroke, Parkinson's disease and Alzheimer's disease, enamel deficiency (eg, enamel hypoplasia), and inappropriate proteolysis (eg, atherosclerosis and abdominal aortic aneurysm) Is useful for potential therapeutic applications involving disorders characterized by For example, a cDNA encoding a tufterin-like protein may be useful in gene therapy to treat enamel hypoplasia and other such disorders, and the tufterin-like protein may be administered to a subject in need thereof. Can be useful if In a non-limiting example, the compositions of the invention have utility in treating patients suffering from disorders of the nervous system. A novel nucleic acid encoding a potassium channel-like protein, and a potassium channel-like protein of the invention, or a fragment thereof, can be used in apocalyptic ataxia type 1, Long QT syndrome 1 and 2, neonatal epilepsy, Gerber- Lange-Nielsen syndrome,
Development of a powerful assay system for the functional analysis of autosomal dominant deafness (DFNA2), non-insulin dependent diabetes mellitus, CNS disorders, arrhythmias, stroke, asthma, hypertension, various disorders that help to understand the pathology of the disease, And may be further useful in the development of new drug targets for various disorders. They can also be used in diagnostic applications, where the presence or amount of nucleic acids or proteins is assessed. These materials are further useful in the production of antibodies that immunospecifically bind to the novel agents of the invention for use in therapeutic or diagnostic methods.

(NOVX Nucleic Acid) The nucleic acid of the present invention contains a nucleic acid encoding a NOVX polypeptide or protein. As used herein, the terms polypeptide and protein are interchangeable.

[0109] In some embodiments, the NOVX nucleic acid encodes a mature NOVX polypeptide. As used herein, the "mature" form of a polypeptide or protein described herein relates to the naturally occurring polypeptide or precursor form or product of a proprotein. Naturally occurring polypeptides, precursors or proproteins include, by way of non-limiting example, the full-length gene product encoded by the corresponding gene. Alternatively, it may be defined as a polypeptide, precursor or proprotein encoded by the open reading frame described herein. The "mature" form of the product occurs, as a further, non-limiting illustration, as a result of one or more naturally occurring processing steps that may occur in the cell in which the gene product is produced. Examples of such processing steps that result in a "mature" form of a polypeptide or protein include cleavage of the N-terminal methionine residue, or proteolysis of the signal peptide or leader sequence, encoded by the start codon of the open reading frame. Examples include sexual amputation. Thus, the mature form resulting from a precursor polypeptide or protein with residues 1-N, where residue 1 is the N-terminal methionine, is the residue 2 that remains after removal of the N-terminal methionine. ~ N
It has an end. Alternatively, the mature form resulting from a precursor polypeptide or protein having residues 1-N (where the N-terminal signal sequence of residues 1-residue M is cleaved) is the remaining residues M + 1-residues It has residues from N. As further used herein, the "mature" form of a polypeptide or protein may result from a post-translational modification step other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation, or phosphorylation. In general, a mature polypeptide or protein may be obtained from the action of only one of these processes, or any combination thereof.

NOVX nucleic acids are among others SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15
, 17, 19 or 21, or a fragment thereof. Further, in the present invention, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 1
5, 17, 19, or 21 mutant or modified nucleic acids or fragments thereof, any of these bases may be represented by SEQ ID NOs: 1, 3, 5, 7, 9,
At least one of NOVX-like activity and physiological function (ie, regulation of angiogenesis, neuronal development), which may be altered from the corresponding base shown at 11, 13, 15, 17, 19, or 21. It still encodes the protein that it maintains. In the present invention, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
Or the complement of 21 nucleic acid sequences, including fragments, derivatives, analogs and homologs thereof. The invention further includes nucleic acids or nucleic acid fragments or their complements whose structure comprises a chemical modification.

One aspect of the invention pertains to isolated nucleic acid molecules that encode NOVX proteins or biologically active portions thereof. Also used as a nucleic acid fragment sufficient for use as a hybridization probe to identify a nucleic acid encoding NOVX (eg, NOVX mRNA) and as a polymerase chain reaction (PCR) primer for amplification or mutation of a NOVX nucleic acid molecule. Also included are fragments for doing so. As used herein, the term "nucleic acid molecule" refers to a DNA molecule (eg, cDNA or genomic DNA), RNA.
Molecules (eg mRNA), DNA produced using nucleotide analogs
Or it is intended to include analogs of RNA, and their derivatives, fragments and homologs. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.

“Probe” refers to nucleic acid sequences of various lengths, preferably at least about 10 nucleotides (nt), 100 nt, or for example about 6 depending on the application.
It is in the range of about 1,000 nt. The probe is used for the detection of identical, similar or complementary nucleic acid sequences. Longer probes are usually obtained from natural or recombinant sources, are highly specific, and hybridize much slower than oligomers. Probes can be single-stranded or double-stranded and designed to have specificity in PCR, membrane-based hybridization techniques or ELISA-like techniques.

An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of this nucleic acid. Examples of isolated nucleic acid molecules include recombinant DNA molecules contained in vectors, recombinant DNA maintained in heterologous host cells.
Molecule, partially or substantially purified nucleic acid molecule, and synthetic DNA or RN
Molecule A, but is not limited thereto. Preferably, an "isolated" nucleic acid has the sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which it is derived (ie, the sequences located at the 5'and 3'ends of the nucleic acid). Not included. For example, in various embodiments, an isolated NOVX nucleic acid molecule has about 50 kb, 25 kb naturally flanking this nucleic acid in the genomic DNA of the cell from which it is derived.
It may comprise less than 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence. In addition, an "isolated" nucleic acid molecule, eg,
A cDNA molecule is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. It may not be included.

Nucleic acid molecules of the invention, eg SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15
, 17, 19, or 21 nucleotide sequences, or the complements of any of these nucleotide sequences, are isolated using standard molecular biology techniques and the sequence information provided herein. Can be separated. SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 as hybridization probes
NOVX nucleic acid sequences can be prepared using standard hybridization and cloning techniques (eg, Sambro).
ok et al. (ed.), MOLECULAR CLONING: A LABORATOR
YY MANUAL 2nd Edition, Cold Spring Harbor Lab
oratory Press, Cold Spring Harbor, NY,
1989; and Ausubel et al., (Eds.), CURRENT PROTOCO.
LS IN MOLECULAR BIOLOGY, John Wiley & S
ons, New York, NY, 1993).

Nucleic acids of the invention are labeled as cDs as templates according to standard PCR amplification techniques.
It can be amplified using NA, mRNA or genomic DNA, and appropriate oligonucleotide primers. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, NO
Oligonucleotides corresponding to VX nucleotide sequences can be prepared by standard synthetic techniques, eg, using an automated DNA synthesizer.

The term “oligonucleotide” as used herein refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases for use in a PCR reaction. Short oligonucleotide sequences can be based on, or designed from, genomic or cDNA sequences, and can be the same, similar or complementary DNA or RNA in a particular cell or tissue.
Is used to amplify, confirm, or reveal its presence. Oligonucleotides include portions of nucleic acid sequences having a length of about 10 nt, 50 nt, or 100 nt, preferably about 15 nt to 30 nt. In one embodiment, the oligonucleotide comprising a nucleic acid molecule of less than 100 nt further comprises SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21.
Of at least 6 consecutive nucleotides, or its complement. Oligonucleotides can be chemically synthesized and used as probes.

In another embodiment, the isolated nucleic acid molecule of the invention is SEQ ID NO: 1, 3, 5, 7
, 9, 11, 13, 15, 17, 19, or 21 of the nucleotide sequence shown as a complement or a nucleic acid molecule that is a part of this nucleotide sequence. Nucleic acid molecules that are complementary to the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21 are SEQ ID NOs: 1, 3, 5, 7, 9, 11
, 13, 15, 17, 19, or 21 is a nucleic acid molecule that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 1, and
It may hydrogen bond to the nucleic acid sequence shown in 7, 19, or 21 with little or no mismatch, thereby forming a stable duplex.

As used herein, the term "complementary" refers to the Wa between nucleotide units of a nucleic acid molecule.
Refers to tson-Crick or Hoogsteen base pairing, and the term "binding" means a physical or chemical interaction between two polypeptides or compounds or related polypeptides or compounds or combinations thereof. Binding includes ionic, nonionic, van der Waals, hydrophobic interactions and the like.
Physical interactions can be direct or indirect. Indirect interactions are
It may be via, or due to, another polypeptide or compound. Direct binding refers to interactions that do not occur through or due to the effects of another polypeptide or compound, but instead are free of other substantial chemical intermediates.

Further, the nucleic acid molecule of the present invention has SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 1.
Only a portion of the 5, 17, 19 or 21 nucleic acid sequence may be included (eg, a fragment that can be used as a probe or primer, or a fragment that encodes the biologically active portion of NOVX). The fragments provided herein provide for at least 6 (consecutive) nucleic acid sequences or at least 4 (consecutive) amino acid sequences, each in the case of nucleic acids allowing specific hybridization, Or, in the case of amino acids, it is defined as (length sufficient to allow specific recognition of the epitope) and is at most some portion less than the full length sequence. Fragments can be derived from any contiguous portion of the selected nucleic acid or amino acid sequences. Derivatives are nucleic acid or amino acid sequences formed from a native compound either directly or by modification or partial substitution. An analog is a nucleic acid sequence or amino acid sequence that has a structure (but not identical) similar to that of the native compound, but differs with respect to a particular component or side chain. Analogs can be synthetic or they can be derived from evolutionarily different sources and have similar or opposite metabolic activity as compared to wild type.

Derivatives and analogs can be full length, or other than full length, if the derivative or analog comprises a modified nucleic acid or amino acid, as described below. A derivative or analog of a nucleic acid or protein of the invention, in various embodiments, when compared to a nucleic acid or protein of the invention, across a nucleic acid or amino acid sequence of identical size, or to an aligned sequence (the alignment is Performed by computer homology programs known in the art), at least about 70%, 80%, 85%, 90%, 95%, 98%, or even 99% identity (preferred identity is between 80 and 80%). 99%) or is substantially homologous thereto, or its encoding nucleic acid hybridizes to the complement of a sequence encoding the above protein under stringent, moderately stringent, or low stringent conditions. Includes, but is not limited to, molecules that include regions. For example, Ausubel et al., CURRENT PROTOCOLS IN M.
OLECULAR BIOLOGY, John Wiley & Sons, N
See ew York, NY, 1993, and below. The illustrated program is a Gap program (Wisconsin Sequence Analysis).
is Package, Version 8 for UNIX (registered trademark),
Genetics Computer Group, University R
essearch Park, Madison, WI) (using default settings,
This is the algorithm of Smith and Waterman (Adv. Appl.
． Math. , 1981, 2: 482-489, which are incorporated herein by reference in their entirety)).

“Homologous nucleic acid sequence” or “homologous amino acid sequence”, or variants thereof, refer to sequences characterized by homology at the nucleotide or amino acid level, as discussed above. The homologous nucleotide sequence is NOVX.
Encodes a sequence encoding an isoform of a polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, the isoforms can be encoded by different genes. In the present invention, homologous nucleotide sequences include species other than humans (including, but not limited to, mammals), such as mouse, rat, rabbit, dog, cat, cow, horse and other organisms. Can be mentioned) NOV
It comprises a nucleotide sequence encoding an X polypeptide. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variants and variants of the nucleotide sequences described herein. However, a homologous nucleotide sequence does not include the nucleotide sequence encoding the human NOVX protein. The homologous nucleic acid sequences are shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18.
, 20 or 22 conservative amino acid substitutions (see below), and NOVX
It includes a nucleic acid sequence encoding a polypeptide having activity. The biological activity of NOVX proteins is described below. The homologous amino acid sequence does not encode the amino acid sequence of human NOVX polypeptide.

Nucleotide sequences determined from cloning of the human NOVX gene have been used to identify and / or clone NOVX homologs in other cell types (eg, from other tissues), as well as NOVX homologs from other mammals. Allows the production of probes and primers designed for use. The probe / primer typically comprises a substantially purified oligonucleotide.
This oligonucleotide is typically represented by SEQ ID NOs: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19 or 21 of at least about 12, about 25, about 50
Pieces, about 100 pieces, about 150 pieces, about 200 pieces, about 250 pieces, about 300 pieces, about 350 pieces
Or about 400 or more contiguous sense strand nucleotide sequences; or SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21 antisense strand nucleotide sequences; or SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 1
At least about 12, about 25, about 50, about 100, about 150, about 200, about 250, about 30 of 5, 17, 19 or 21 naturally occurring variants.
It contains regions of nucleotide sequences that hybridize to 0, about 350, or about 400 or more contiguous sense strand nucleotide sequences under stringent conditions.

Probes based on human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a labeling group attached to the probe, eg, the labeling group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such a probe may, for example, measure the level of nucleic acid encoding NOVX in a sample of cells from a subject as part of a diagnostic test kit for identifying cells or tissues that misexpress NOVX protein. (Eg, detecting NOVX mRNA levels, or determining whether the genomic NOVX gene is mutated or deleted).

A “polypeptide having a biologically active portion of NOVX” refers to a polypeptide having an activity similar to, but not necessarily identical to, that of the polypeptide of the present invention, with or without dose dependence. Instead, it includes the mature form as measured in certain biological assays. A nucleic acid fragment encoding a "NOVX biologically active portion" is SEQ ID NO: that encodes a polypeptide having NOVX biological activity (NOVX protein biological activity is described below). 1, 3,
5,7,9,11,13,15,17,19 or part of 21 was isolated and NO
It can be prepared by expressing the encoded portion of the VX protein (eg, by recombinant expression in vitro) and assessing the activity of the encoded portion of NOVX. For example, a nucleic acid fragment encoding a biologically active portion of NOVX can optionally include an ATP binding domain. In another embodiment, the nucleic acid fragment encoding the biologically active portion of NOVX is 1
Including one or more areas.

Variants of NOVX The present invention further comprises SEQ ID NOs: 1, 3, 5, 7, 9 due to the degeneracy of the genetic code.
, 11, 13, 15, 17, 19 or 21 are included in the nucleic acid molecule. Thereby, these nucleic acids are
, 7, 9, 11, 13, 15, 17, 19 or 21 is a protein encoded by the nucleotide sequence (eg, SEQ ID NOs: 2, 4, 6, 8, 10).
, 12, 14, 16, 18, 20 or 22)) and encodes the same NOVX protein. In another embodiment, the isolated nucleic acid molecule of the invention encodes a protein having the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22. Has a nucleotide sequence that

In addition to the human NOVX nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21, DNA sequence polymorphisms leading to changes in the amino acid sequence of NOVX are It will be appreciated by those of skill in the art that they may exist within a population (eg, a human population). Such genetic polymorphisms in the NOVX gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to N
Refers to a nucleic acid molecule containing an open reading frame encoding an OVX protein, preferably a mammalian NOVX protein. Such natural allelic variations can typically result in 1-5% variability in the nucleotide sequence of the NOVX gene. Is the result of natural allelic variation,
And any and all such nucleotide variations within NOVX and resulting amino acid polymorphisms that do not alter the functional activity of NOVX are intended to be within the scope of the present invention.

In addition, it encodes NOVX proteins from other species, thus SEQ ID NO: 1,
Nucleic acid molecules having a nucleotide sequence that differs from the human sequence of 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21 are intended to be within the scope of the present invention. Nucleic acid molecules corresponding to naturally occurring allelic variants and homologs of the NOVX cDNAs of the invention are based on their homology to the human NOVX nucleic acids disclosed herein under standard stringent hybridization conditions. Can be isolated using human cDNA or a portion thereof as a hybridization probe according to conventional hybridization techniques. For example, soluble human NOVX
cDNA can be isolated based on its homology to human membrane-bound NOVX. Similarly, membrane-bound human NOVX cDNA can be isolated based on its homology to soluble human NOVX.

Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and, under stringent conditions, SEQ ID NOs: 1, 3, 5, 7
, 9, 11, 13, 15, 17, 19 or 21 nucleotide sequences. In another embodiment, the nucleic acid is at least 10
, 25, 50, 100, 250, 500 or 750 nucleotides long. In another embodiment, the isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term “hybridize under stringent conditions” refers to conditions under which hybridization, and washing, nucleotide sequences that are at least 60% homologous to one another typically remain hybridized to each other. Intended to be listed.

Homologs (ie, nucleic acids encoding NOVX proteins from non-human species) or other related sequences (eg, paralogs) are probed with all or part of a particular human sequence and are labeled with nucleic acid hybrids. It can be obtained by low, medium, or high stringency hybridization using methods well known in the art for hybridization and cloning.

As used herein, the phrase “stringent hybridization conditions” refers to under which a probe, primer or oligonucleotide is
It is a condition that hybridizes to the target sequence but does not hybridize to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 ° C. below the thermal melting point (T _m ) of a particular sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probe complementary to the target sequence hybridizes to the target sequence at equilibrium. Since the target sequence is generally present in excess, at Tm 50% of the probe is occupied in equilibrium. Typically, stringent conditions are pH 7.0-8.3 and salt concentrations less than about 1.0 M sodium ion, typically about 0.01-1.0.
M sodium ion (or other salt), and temperature at least about 30 ° C. for short probes, primers or oligonucleotides (eg 10 nt to 50 nt) and at least about 60 ° C. for longer probes, primers and oligonucleotides. It is a condition. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

Stringent conditions are known to those of skill in the art, and may be CURRENT PR
OTOCOLS IN MOLECULAR BIOLOGY, John Wi
ley & Sons, N.M. Y. (1989), 6.3.1-6.3.6. Preferably, this condition is such that sequences that are at least about 65%, about 70%, about 75%, about 85%, about 90%, about 95%, about 98% or about 99% homologous to each other, typically to each other. Conditions are such that they will remain hybridized. A non-limiting example of stringent hybridization conditions is 6 × SSC, 50 mM
Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP,
Hybridization at 65 ° C. in high salt buffer containing 0.02% Ficoll, 0.02% BSA, and 500 mg / ml denatured salmon sperm DNA. This hybridization is followed by one or more washes at 50 ° C. in 0.2 × SSC, 0.01% BSA. SEQ ID NOs: 1, 3 under stringent conditions
An isolated nucleic acid molecule of the invention which hybridizes to the sequence 5, 7, 9, 11, 13, 15, 17, 19 or 21 corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule is an R that has a naturally-occurring (eg, encoding a native protein) nucleotide sequence.
Refers to NA or DNA molecules.

In a second embodiment, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17
, 19 or 21, or a fragment, analog or derivative thereof, is provided with a nucleic acid sequence capable of hybridizing under moderate stringency conditions to a nucleic acid molecule. A non-limiting example of moderate stringency hybridization conditions is hybridization at 55 ° C. in 6 × SSC, 5 × Denhardt's solution, 0.5% SDS and 100 mg / ml denatured salmon sperm DNA, followed by One or more washes at 37 ° C. in 1 × SSC, 0.1% SDS. Other conditions of moderate stringency that can be used are well known in the art. For example, Ausubel et al. (Eds.), 1993, CURRE.
NT PROTOCOLS IN MOLECULAR BIOLOGY, Jo
hn Wiley & Sons, NY and Kriegler, 1990, G.
ENE TRANSFER AND EXPRESSION, A LABORA
See TORY MANUAL, Stockton Press, NY.

In a third embodiment, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17
, 19 or 21, or a fragment, analog or derivative thereof, a nucleic acid molecule capable of hybridizing under low stringency conditions to a nucleic acid molecule. A non-limiting example of low stringency hybridization conditions is 35% formamide, 5 × SSC, 50 mM Tris.
-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02%
Ficoll, 0.2% BSA, 100 mg / ml denatured salmon sperm DNA,
Hybridization in 10% (weight / volume) dextran sulfate at 40 ° C., followed by 2 × SSC, 25 mM Tris-HCl (pH 7.4), 5 mM.
One or more washes at 50 ° C. in EDTA and 0.1% SDS. Other conditions of low stringency that can be used are well known in the art (eg,
As used for cross-species hybridization). For example, Ausu
bel et al. (ed.), 1993, CURRENT PROTOCOLS IN MO.
LECULAR BIOLOGY, John Wiley & Sons, NY
And Kriegler, 1990, GENE TRANSFER AND
EXPRESSION, A LABORATORY MANUAL, Stock
ton Press, NY; Shilo and Weinberg, 1981, P.
See roc Natl Acad Sci USA 78: 6789-6792.

Conservative Mutations In addition to naturally occurring allelic variants of NOVX sequences that may be present in the population, one of skill in the art would appreciate that SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, Changes can be introduced by mutations to the nucleotide sequence of 15, 17, 19 or 21 thereby encoding the encoded NOV without altering the functional capacity of the NOVX protein.
It is further understood that changes are made to the amino acid sequence of the X protein. For example, a nucleotide substitution that results in an amino acid substitution at a "non-essential" amino acid residue is:
It can be performed in the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21. A "non-essential" amino acid residue is a residue that can be altered from the NOVX wild-type sequence without significantly altering its biological activity, while "
The "essential" amino acid residues are required for biological activity. For example, the NOV of the present invention
Amino acid residues conserved among X proteins are predicted to be particularly unacceptable for alteration.

Another aspect of the invention includes changes in amino acid residues that are not essential for activity,
It relates to nucleic acid molecules encoding NOVX proteins. Such NOVX proteins have amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,
Unlike 18, 20, or 22, it still retains biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence that encodes a protein, wherein the protein is SEQ ID NO: 2, 4, 6, 8, 1 respectively.
It comprises an amino acid sequence that is at least about 75% homologous to the 0, 12, 14, 16, 18, 20, or 22 amino acid sequence. Preferably, the protein encoded by this nucleic acid molecule is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 1.
At least about 80% homology to 6, 18, 20, or 22 and more preferably at least about 90% to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22. , About 95%, about 98% homology, and most preferably at least about 99% homology.

An isolated nucleic acid molecule encoding a NOVX protein that is homologous to the protein is
It can be produced by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21 and, as a result, One or more amino acid substitutions, additions or deletions are introduced into the encoded protein.

Mutations are standard techniques (eg, site-directed mutagenesis and PCR-mediated mutagenesis).
Can be introduced into the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21. Preferably, the conservative amino acid substitution is
Made with one or more putative nonessential amino acid residues. "Conservative amino acid substitution" means
An amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. A family of amino acid residues with similar side chains is defined in the art. These families include: amino acids with basic side chains (eg lysine, arginine, histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains. Amino acids (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side Amino acids with chains (eg threonine, valine, isoleucine) and amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in NOVX is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, the mutation is along all or part of the NOVX coding sequence (eg, saturation mutagenesis).
can be randomly introduced (by is) and the resulting mutants can be screened for biological activity of NOVX to identify those that retain the activity. SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21
Following mutagenesis, the encoded protein can be expressed by any recombinant technique known in the art and the activity of the protein can be determined.

In one embodiment, mutant NOVX proteins can be assayed for: (1) other NOVX proteins, other cell surface proteins, or biologically active portions thereof, and protein: protein interactions. The ability to form an action,
(2) Complex formation between mutant NOVX protein and NOVX receptor;
(3) Mutant NOVX protein binds to intracellular target protein or biologically active portion thereof; (eg, avidin protein); (4) Ability to bind NOVX protein; or (5) Anti-NOVX protein antibody The ability to specifically bind to.

(Antisense NOVX Nucleic Acid) Another aspect of the present invention is SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 15, 17.
, 19 or 21, or a fragment, analog or derivative thereof, to an isolated antisense nucleic acid molecule capable of hybridizing to or complementary to a nucleic acid molecule. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid that encodes a protein (eg, complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). . In particular aspects, at least about 10, about 25, about 50, about 100, about 25
Antisense nucleic acid molecules are provided that comprise sequences complementary to 0 or about 500 nucleotides or the entire NOVX coding strand, or only a portion thereof. NO of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22
Nucleic acid molecules encoding fragments, homologues, derivatives and analogs of VX protein, or SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 17, 19
Alternatively, an antisense nucleic acid complementary to the nucleic acid sequence of 21 NOVX is further provided.

In one embodiment, the antisense nucleic acid molecule is antisense to the “coding region” of the coding strand of the nucleotide sequence encoding NOVX. The term "coding region" refers to a region of a nucleotide sequence that contains codons translated into amino acid residues (eg, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18).
, 20 or 22, corresponding to the protein coding region of human NOVX). In another embodiment, the antisense nucleic acid molecule is antisense to the “non-coding region” of the coding strand of the nucleotide sequence encoding NOVX. The term “non-coding region” refers to the 5 ′ and 3 ′ sequences that are flanked by coding regions and are not translated into amino acids (ie, also referred to as 5 ′ untranslated region and 3 ′ untranslated region).

Considering the coding strand sequences encoding NOVX disclosed herein (eg, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21), Antisense nucleic acids of the invention include Watson and Crick or Ho.
It can be designed according to the rules of base pairing of ogsteen. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of NOVX mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA.
Antisense oligonucleotides can be, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45 or about 50 nucleotides in length. The antisense nucleic acids of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, antisense nucleic acids (eg, antisense oligonucleotides) are formed to naturally occur nucleotides, or to increase the biological stability of this molecule, or between antisense and sense nucleic acids. It can be chemically synthesized using variously modified nucleotides designed to increase the physical stability of the duplex (eg,
Phosphorothioate derivatives and acridine substituted nucleotides can be used)
.

Examples of modified nucleotides that can be used to make antisense nucleic acids include: 5-fluorouracil, 5-bromouracil, 5-
Chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β -D-galactosyleosin (galact)
osylqueosine), inosine, N6-isopentenyladenine, 1-
Methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N
6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyleosin (ma)
nnosylqueosine), 5′-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wibutoxin, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,
2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-
Thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be biologically produced using an expression vector in which the nucleic acid has been subcloned in the antisense orientation (ie, RNA transcribed from the inserted nucleic acid.
Is in the antisense orientation with respect to the target nucleic acid of interest, as further described in the subsection below).

The antisense nucleic acid molecules of the invention are typically administered to a subject or produced in situ so that they hybridize to the cellular mRNA and / or genomic DNA encoding the NOVX protein. Soybeans or binds thereto, thereby inhibiting the expression of this protein, for example by inhibiting transcription and / or translation. Hybridization may be by conventional nucleotide complementation to form a stable duplex, or in the case of antisense nucleic acid molecules that bind to a DNA duplex, for example, in the double helix major group (
via a specific interaction in major groove). Examples of routes of administration of the antisense nucleic acid molecules of the invention include direct injection at the tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified so that they specifically bind to receptors or antigens expressed on the surface of selected cells. This is, for example, by linking the antisense nucleic acid molecule to a peptide or antibody that binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. Vector constructs in which the antisense nucleic acid molecule is placed under the control of the strong pol II or pol III promoter in order to achieve a sufficient intracellular concentration of the antisense molecule are preferred.

In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. The α-anomeric nucleic acid molecule forms a specific double-stranded hybrid with complementary RNA. Here, in contrast to the normal β-units, the chains run parallel to each other (Gaultier et al. (1987) Nucleic Acids.
Res 15: 6625-6641). This antisense nucleic acid molecule also contains 2
'-O-Methylribonucleotides (Inoue et al., (1987) Nucleic
Acids Res 15: 6131-6148) or chimeric RNA-DN
A analog (Inoue et al. (1987) FEBS Lett 215: 327-
330).

Such modifications include, by way of non-limiting example, modified bases and nucleic acids with modified or derivatized sugar phosphate backbones. These modifications are performed, at least in part, to enhance the chemical stability of the modified nucleic acids so that they are used as antisense binding nucleic acids, eg, in therapeutic applications in a subject. obtain.

NOVX Ribozymes and PNA Moieties In yet another embodiment, the antisense nucleic acids of the invention are ribozymes.
Ribozymes are catalytic RNA molecules having ribonuclease activity that can cleave single-stranded nucleic acids (eg, mRNA), and these have a complementary region to the single-stranded nucleic acid. Therefore, ribozymes (for example, hammerhead ribozyme (H
aselhoff and Gerlach (1988) Nature 334: 5.
85-591)) can be used to catalytically cleave NOVX mRNA transcripts, thereby inhibiting translation of NOVX mRNA. Ribozymes having specificity for nucleic acids encoding NOVX are disclosed in NOVX
The nucleotide sequence of DNA (ie, SEQ ID NOs: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19 or 21). For example, derivatives of Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence that is cleaved within the mRNA encoding NOVX. See, eg, Cech et al., US Pat. No. 4,987,071; and Cech et al., US Pat. No. 5,116,742. Or NO
VX mRNA can be used to select catalytic RNAs with specific ribonuclease activity from a pool of RNA molecules. For example, Bartel et al. (1993)
See Science 261: 1411-1418.

Alternatively, NOVX gene expression targets a nucleotide sequence complementary to the regulatory regions of NOVX (eg, the NOVX promoter and / or enhancer) and creates a triple helix structure that interferes with transcription of the NOVX gene in target cells. Can be inhibited by forming. Generally, Helene. (1991) Antic
cancer Drug Des. 6: 569-84; Helene et al. (1992).
) Ann. N. Y. Acad. Sci. 660: 27-36; and Maher
(1992) Bioassays 14: 807-15.

In various embodiments, NOVX nucleic acids may be modified at the base, sugar, or phosphate backbones to improve, for example, the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of nucleic acids can be modified to produce peptide nucleic acids (Hyrup et al. (1996) Bioorg Med C.
hem 4: 5-23). As used herein, the term "peptide nucleic acid" or "PNA" replaces the deoxyribose phosphate backbone with a pseudopeptide backbone and retains only the four natural nucleobases. Refers to nucleic acid mimics (eg, DNA mimics). The neutral backbone of PNA has been shown to allow specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomer is
Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996).
It can be performed using standard solid phase peptide synthesis protocols as described in PNAS 93: 14670-675.

NOVX PNAs can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific regulation of gene expression, for example by inducing transcription or translation arrest or inhibiting replication. NOVX PNAs also include, for example, PNA directional PCs.
In the analysis of single base pair mutations in genes by R-clamping; other enzymes (
For example, as an artificial restriction enzyme when used in combination with S1 nuclease (Hyrup B. (1996) supra); or as a probe or primer for DNA sequences and hybridization (Hyrup et al. (1996).
Above; Perry-O'Keefe (1996), supra), may be used.

In another embodiment, NOVX PNAs are labeled with PNA-DNA chimeras, eg, by attaching lipophilic or other helper groups to the PNAs to enhance their stability or cellular uptake. Or by the use of liposomes or other techniques of drug delivery known in the art. For example, NOVX PNAs that may combine the advantageous properties of PNAs and DNA.
-A DNA chimera can be generated. While the PNA moiety provides high binding affinity and specificity, such chimeras have DNA recognition enzymes (eg, RNase H
And DNA polymerase) to interact with the DNA portion. PN
A-DNA chimeras can be ligated using a linker of appropriate length selected with consideration of stacking of bases, the number and orientation of bonds between nucleobases (Hyrup (
1996) above). The synthesis of PNA-DNA chimeras is described in Hyrup (1996) supra and Finn et al. (1996) Nucl Acids Res 24: 335.
It can be performed as described in 7-63. For example, DNA strands can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs such as 5 '-(4-methoxytrityl).
Amino-5'-deoxy-thymidine phosphoramidites) can be used between the PNA and the 5'end of DNA (Mag et al. (1989) Nucl Acid R).
es 17: 5973-88). The PNA monomers are then coupled in a stepwise fashion to produce a chimeric molecule having a 5'PNA segment and a 3'DNA segment (Finn et al. (1996) supra). Alternatively, a 5'DNA segment and a 3'PNA segment can be used to synthesize a chimeric molecule. Pete
rsen et al. (1975) Bioorg Med Chem Lett 5:11.
19-11124.

In other embodiments, the oligonucleotide may include other accessory groups such as: peptides (eg, for targeting host cell receptors in vivo), or transport across cell membranes. Factors (eg, Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:65.
53-6556; Lemaitre et al., 1987, Proc. Natl. Aca
d. Sci. 84: 648-652; PCT Publication No. WO88 / 09810), or the blood-brain barrier (eg PCT Publication No. WO89 / 10134).
checking). In addition, the oligonucleotide may be conjugated to a hybridization-triggered cleavage agent (eg, Krol et al., 1988, BioTechniques 6: 9).
58-976), or an intercalating agent (e.g., Zon, 1).
988, Pharm. Res. 5: 539-549)). For this purpose, oligonucleotides are different molecules (eg peptides,
Hybridization-induced cross-linking agents, transport agents, hybridization-induced cleavage agents, etc.).

(NOVX Polypeptide) The NOVX polypeptide of the present invention has a sequence of SEQ ID NOs: 2, 4, 6, 8, and 10.
, 12, 14, 16, 18, 20 or 22 provided with a NOVX-like protein. The present invention also encodes a protein, or a functional fragment thereof, which still retains its NOVX-like activity and physiological function, any residue of which is SEQ ID NO: 2, 4, 6, 8, 10, 12, 14. , 16, 18, 20 or 22 which may vary from the corresponding residue. In some embodiments, in the variant or variant protein, 20
Up to% or more residues may be so altered. In some embodiments, the NOVX polypeptides according to the present invention are mature polypeptides.

Generally, a NOVX-like variant that retains a NOVX-like function has a residue at a particular position in the sequence replaced by another amino acid, and additionally has an additional residue inserted between the two residues of the parent protein. And the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion or deletion
Included in the present invention. In the preferred situation, this substitution is a conservative substitution as defined above.

One aspect of the invention relates to an isolated NOVX protein, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. Polypeptide fragments suitable for use as an immunogen to raise anti-NOVX antibodies are also provided. In one embodiment, native NOV
The X protein can be isolated from the cell or tissue source by a suitable purification scheme using standard protein purification techniques. In another embodiment, the NOVX protein is produced by recombinant DNA technology. As an alternative to recombinant expression, N
OVX proteins or polypeptides can be chemically synthesized using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins derived from the cell or tissue source from which the NOVX protein is derived. Or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes a preparation of NOVX protein, in which preparation
From the cellular components of cells in which the NOVX protein is isolated or recombinantly produced, NO
VX protein is isolated. In one embodiment, the term "substantially free of cellular material" refers to less than about 30% (by dry weight) of non-NOVX proteins (also referred to herein as "contaminant proteins"), more preferably. Include preparations of NOVX proteins having less than about 20% non-NOVX proteins, even more preferably less than about 10% non-NOVX proteins, and most preferably less than about 5% non-NOVX proteins. If the NOVX protein or biologically active portion thereof is recombinantly produced, preferably the preparation is also substantially free of culture medium. That is, the culture medium is about 2 times the volume of the protein preparation.
It represents less than 0%, more preferably less than about 10%, and most preferably less than about 5%.

The term “substantially free of chemical precursors or other chemicals” refers to a preparation of NOVX proteins in which the protein is separated from the chemical precursors or other chemicals involved in the synthesis of that protein. including. In one embodiment, the term "substantially free of chemical precursors or other chemicals" refers to chemical precursors or non-NO
Less than about 30% VX chemistry (by dry weight), more preferably less than about 20% chemical precursor or non-NOVX chemistry, even more preferably less than about 10% chemical precursor or non-NOVX chemistry, And most preferably a chemical precursor or non-N
Included are preparations of NOVX protein having less than about 5% OVX chemistry.

The biologically active portion of the NOVX protein comprises fewer amino acids than the full-length NOVX protein and exhibits at least one of the activities of the NOVX protein (eg, SEQ ID NOs: 2, 4, 6, 8, 1
(Amino acid sequence shown in 0, 12, 14, 16, 18, 20 or 22) or a peptide containing an amino acid sequence derived from the amino acid sequence of NOVX protein. Typically, the biologically active moiety is NO
It comprises at least one active domain or motif of the VX protein. The biologically active portion of the NOVX protein has, for example, a length of 10, 25,
It can be a polypeptide that is 50, 100 or more amino acids.

The biologically active portion of the NOVX proteins of the invention may include at least one of the above identified domains (eg, TSR modules) that are conserved between NOVX proteins. In addition, other biologically active portions in which other regions of the protein have been deleted can be prepared by recombinant techniques, and native NO
It can be evaluated for one or more of the functional activities of the VX protein.

[0159] In one embodiment, the NOVX protein has SEQ ID NOs: 2, 4, 6, 8,
It has the amino acid sequence shown in 10, 12, 14, 16, 18, 20 or 22. In other embodiments, the NOVX protein is SEQ ID NO: 2, 4, 6, 8,
Although substantially homologous to 10, 12, 14, 16, 18, 20, or 22 and differing in amino acid sequence due to natural allelic variants or mutagenesis, as discussed in detail below. , SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 1
It retains the functional activity of 6, 18, 20 or 22 proteins. Thus, in another embodiment, the NOVX protein has SEQ ID NOs: 2, 4, 6, 8, 10, 1,
2, 14, 16, 18, 20, or 22, containing at least about 45% homology to the amino acid sequence, and SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14.
, 16, 18, 20 or 22 NOVX proteins that retain the functional activity.

Determining Homology Between Two or More Sequences To determine the percentage of homology between two amino acid sequences or two nucleic acids, the sequences are aligned for the purpose of optimal comparison. (For example, a gap can be introduced in any of the sequences that are compared for optimal alignment between the sequences). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (ie, as used herein). , "Homology" of amino acids or nucleic acids is equivalent to "identity" of amino acids or nucleic acids).

Nucleic acid sequence homology can be determined as the degree of identity between two sequences. Homology can be determined using computer programs known in the art, such as GAP software provided in the GCG program package. Needleman and Wunsch 1970 J Mol Biol
48: 443-453. The following settings for comparing nucleic acid sequences (GA
Using GCG GAP software with a P production penalty of 5.0, and a GAP extension penalty of 0.3), the coding regions for similar nucleic acid sequences referred to above are SEQ ID NOs: 1, 3, 5, 7 , 9, 11, 13, 15, 17, 19
Or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the CDS (coding) portion of the DNA sequence shown in 21 or 21. Indicates the degree of sex.

The term “sequence identity” refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term “percentage of sequence identity” is calculated by: comparing two sequences that are optimally aligned over this region of comparison, the same nucleobase in both sequences (eg A in the case of nucleic acids). , T, C, G, U, or I) determines the number of positions that are present and derives the number of matched positions;
Dividing by the total number of positions within the comparison region (ie, window size), and multiplying the result by 100 yields the percentage sequence identity. The term "substantial identity" as used herein refers to a characteristic of a polynucleotide sequence, where the polynucleotide has at least 80% of the sequence compared to the reference sequence over the comparison region. Identity, preferably at least 85% sequence identity, and often 90-95% sequence identity, more usually at least 99%.
Includes sequences with% sequence identity. The term "percentage of positive residues" is calculated by the following: comparing two sequences that are optimally aligned over their comparison region, identical amino acids and conservative amino acid substitutions as described above. Determining the number of positions present in both sequences and deriving the number of matching positions, the number of matching positions is determined by the total number of positions in the comparison region (ie,
Window size) and multiply the result by 100 to derive the percentage of positive residues.

Chimeric and Fusion Proteins The present invention also provides NOVX chimeric or fusion proteins.
As used herein, a NOVX "chimeric protein" or NOVX "
A "fusion protein" is a NOV operably linked to a non-NOVX polypeptide.
X polypeptides. “NOVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to NOVX, whereas “non-NOVX polypeptide” refers to NO
Proteins that are not substantially homologous to VX proteins (eg, proteins that differ from NOVX proteins and are from the same or different organisms)
Refers to a polypeptide having an amino acid sequence corresponding to. In the NOVX fusion protein, the NOVX polypeptide may correspond to all or a portion of the NOVX protein. In one embodiment, the NOVX fusion protein is NOVX.
It comprises at least one biologically active portion of a protein. In another embodiment, the NOVX fusion protein comprises at least two biologically active portions of NOVX protein. In a fusion protein, the term "operably linked"
Is intended to indicate that the NOVX and non-NOVX polypeptides are fused in-frame to each other. The non-NOVX polypeptide can be fused to the N-terminus or C-terminus of the NOVX polypeptide.

For example, in one embodiment, a NOVX fusion protein comprises a NOVX polypeptide operably linked to the extracellular domain of a second protein. Such fusion proteins can be further utilized in screening assays for compounds that modulate NOVX activity (such assays are described in detail below).

In another embodiment, the fusion protein is a GST-NOVX fusion protein, wherein the NOVX sequences are GST (ie glutathione S-.
Transferase) sequence is fused to the C-terminus. Such fusion proteins may facilitate the purification of recombinant NOVX.

In another embodiment, the fusion protein is a NOVX-immunoglobulin fusion protein, wherein a NOVX sequence containing one or more domains is fused to sequences derived from a member of the immunoglobulin protein family. It This NOVX-immunoglobulin fusion protein of the invention is incorporated into a pharmaceutical composition and administered to a subject to direct NOVX ligand and NOV on the surface of cells.
It may inhibit the interaction with the X protein, thereby suppressing NOVX-mediated signaling in vivo. In one non-limiting example, contemplated NOVX ligands of the invention are NOVX receptors. This NOVX-immunoglobulin fusion protein can be used to influence the bioavailability of NOVX cognate ligands. Inhibition of NOVX ligand / NOVX interactions treats both proliferative and differentiation disorders (eg, cancer) and regulates (eg, promotes or inhibits) cell survival, as well as acute and chronic inflammatory disorders and hypersensitivity disorders. It may be therapeutically useful for plastic wound healing, such as hyperplastic scars and keloids. Further, this NOVX-immunoglobulin fusion protein of the invention can be used as an immunogen to produce anti-NOVX antibodies in a subject, purify NOVX ligand and inhibit NOVX interaction with NOVX ligand. Can be used in a screening assay to identify the molecule that is to be processed.

The NOVX chimeric or fusion proteins of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences may be labeled according to conventional techniques, eg, blunt-ended or cohesive (for ligation).
stagger) ends, restriction enzyme digestion to provide suitable ends, cohesive end fill-ins where necessary, alkaline phosphatase treatment to avoid unwanted ligations, and enzymatic ligation Connect together in a frame. In another embodiment, the fusion gene may be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed with anchor primers that produce complementary overhangs between two consecutive gene fragments that can then be annealed and reamplified to produce a chimeric gene sequence (eg, Ausbel et al. (Edit) CURRENT P
ROTOCOLS IN MOLECULAR BIOLOGY, John W
See iley & Sons, 1992). In addition, fusion components (eg G
Many expression vectors that already encode ST polypeptides) are commercially available.
The nucleic acid encoding NOVX can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.

NOVX Agonists and Antagonists The present invention also relates to variants of NOVX proteins that function either as NOVX agonists (mimetics) or as NOVX antagonists. N
Variants of OVX proteins, such as discrete point mutations or truncations of NOVX proteins, can be generated by mutagenesis. An agonist of a NOVX protein may retain substantially the same biological activity as the naturally occurring form of the NOVX protein, or a subset of that biological activity. An antagonist of a NOVX protein may inhibit one or more of the activities of the naturally occurring form of the NOVX protein, eg, by competitively binding to a downstream or upstream member of the cell signaling cascade that includes the NOVX protein. Thus, a particular biological effect may be elicited by treatment with a variant of limited function. In one embodiment, treatment of the subject with a variant having a subset of biological activities of the naturally occurring form of the protein is performed on the subject relative to treatment with the naturally occurring form of the NOVX protein. Have less side effects in.

Variants of NOVX proteins that function either as NOVX agonists (mimetics) or as NOVX antagonists are combinatorial live mutants of NOVX proteins for NOVX protein agonist or antagonist activity, eg truncated variants. It can be identified by screening the rally. In one embodiment, a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A diverse library of NOVX variants includes, for example, a mixture of synthetic oligonucleotides, a degenerate set of potential NOVX sequences, either as individual polypeptides or within a set of NOVX sequences (eg, It can be produced by enzymatically linking to a gene sequence so that it can be expressed as a larger set of fusion proteins (for phage display). There are a variety of methods that can be used to generate libraries of potential NOVX variants from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed in an automated DNA synthesizer, which synthetic gene is then ligated into an appropriate expression vector. The use of a degenerate set of genes allows for the supply of all of the sequences encoding the desired set of potential NOVX sequences in one mixture. Methods of synthesizing degenerate oligonucleotides are known in the art (eg, Narang (1983) Tetrahedron 39: 3; Itak).
ura et al. (1984) Annu Rev Biochem 53: 323; It.
Akura et al. (1984) Science 198: 1056; Ike et al. (19).
83) Nucl Acid Res 11: 477).

Polypeptide Libraries In addition, libraries of fragments of NOVX protein coding sequences can be used to generate a diverse population of NOVX fragments for screening and subsequent selection of variants of NOVX protein. In one embodiment, the library of coding sequence fragments comprises treating a double-stranded PCR fragment of a NOVX coding sequence with a nuclease under conditions that produce only about one nick per molecule, double-stranded DNA. Denaturing the DNA, regenerating this DNA to form double-stranded DNA that may contain sense / antisense pairs from different nick products, and treating the double stranded DNA by treatment with S1 nuclease It can be generated by removing the single-stranded portion and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes various sized N-terminal and internal fragments of the NOVX protein.

To screen the gene products of combinatorial libraries generated by point mutations or truncations, and the cD of gene products with selected properties
Several techniques for screening NA libraries are known in the art. Such techniques are applicable to rapid screening of gene libraries generated by combinatorial mutagenesis of NOVX proteins.
The most widely used technique suitable for high-throughput analysis for screening large gene libraries is typically by cloning the gene library into a replicable expression vector, in which the resulting library of vectors is Transforming appropriate cells, and expressing the combinatorial gene under conditions under which detection of the desired activity facilitates isolation of the vector encoding the gene for which the product was detected. A novel technique for increasing the frequency of functional variants in the library, Recruitable Ensemble Mutagenesis (REM), can be used in combination with screening assays to identify NOVX variants (Arkin).
And Yourvan (1992) PNAS 89: 7811-7815; De.
lgrave et al. (1993) Protein Engineering 6: 3.
27-331).

[0172]   (NOVX antibody)   Antibody to NOVX protein, or fragment of NOVX protein
Are also included in the present invention. As used herein, the term "antibody" is exempt.
Immunoglobulin molecules, and immunologically active portions of immunoglobulin (Ig) molecules
(Ie, a molecule containing an antigen binding site that specifically binds (immunoreacts with) an antigen
). Such antibodies include polyclonal antibodies and monoclonal antibodies.
, Chimeric antibody, single chain antibody, F_abFragment, F_{ab '}Fragment and F _{(Ab ') 2} Fragment, and F_abAn expression library can be mentioned,
It is not limited to these. Generally, antibody molecules obtained from humans include IgG, Ig
It is associated with any class of M, IgA, IgE and IgD, which are present in the molecule.
They differ depending on the nature of the heavy chain present. Certain classes are also subclasses (eg
For example, IgG₁, IgG_TwoEtc.). Furthermore, in humans, the light chain is κ
It can be a chain or a lambda chain. References herein to antibodies include all human antibody species.
Contains references to all such classes, subclasses and types.

The isolated NOVX-related proteins of the invention can be intended to serve as antigens, or parts or fragments thereof, and further used as immunogens for the preparation of polyclonal and monoclonal antibodies. Antibodies that immunospecifically bind to the antigen can be produced using standard techniques of. Full length proteins can be used, or the invention provides antigenic peptide fragments of the antigen for use as immunogens. The antigenic peptide fragment is an amino acid sequence of a full-length protein (for example, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,
At least 6 amino acid residues of the amino acid sequence shown in 16, 18, 20 or 22) and their epitopes, so that the antibody raised against this peptide is any full-length protein or any epitope containing this epitope. Form specific immune complexes with fragments of Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. The preferred epitopes encompassed by this antigenic peptide are the regions of the protein located on its surface; usually these are hydrophilic regions.

In certain embodiments of the invention at least one encompassed by an antigenic peptide.
One epitope is a region of a NOVX-related protein located on the surface of the NOVX protein, eg, a hydrophilic region. Hydrophobicity analysis of the human NOVX-related protein sequences reveals which regions of the NOVX-related protein are particularly hydrophilic and, therefore, appear to encode useful surface residues for targeting antibody production. Show. As a means of targeting antibody production, hydropathic plots showing hydrophilic and hydrophobic regions, eg, with or without a Fourier transform, Kyte
It may be produced by any method known in the art, including the Doolittle or Hopp Woods method. For example, Hopp and Woods, 1981, Proc., Each of which is incorporated herein by reference in its entirety. Nat. A
cad. Sci. USA 78: 3824-3828; Kyte and Dool.
title 1982, J. Mol. Biol. 157: 105-142. Antibodies specific for one or more domains within the antigenic protein or derivative, fragment, analog or homologue thereof are also provided herein.

The proteins of the invention, or derivatives, fragments, analogs, homologs or orthologs thereof, can be utilized as immunogens in the production of antibodies that immunospecifically bind to these protein components.

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

Polyclonal Antibodies For the production of polyclonal antibodies, a variety of suitable host animals (eg, rabbits, goats, mice or other mammals) may have native proteins, synthetic variants thereof, or derivatives thereof as described above. Can be immunized by one or more injections with. Suitable immunogenic preparations include, for example, a naturally occurring immunogenic protein, a chemically synthesized polypeptide presenting the immunogenic protein, or a recombinantly expressed immunogenic protein. obtain. In addition, this protein can be conjugated to a second protein known to be immunogenic in the immunized mammal. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. This preparation further
An adjuvant may be included. Various adjuvants have been used to increase the immunological response, such as Freund's (complete and incomplete), mineral gels (eg aluminum hydroxide), surfactants (eg lysolecithin, pluronic). (Pluronic) polyols, polyanions, peptides, oil emulsions, dinitrophenols, etc., human-useable adjuvants (eg Bacille Calmette-Guerin and Cory).
nebacterium parvum) or similar immunostimulants, but are not limited thereto. Further examples of adjuvants that can be used include:
MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate).

Polyclonal antibody molecules directed against immunogenic proteins can be isolated from mammals (eg from blood) and are well known in the art (eg affinity chromatography using protein A or protein G (eg. This can be further purified mainly by providing the IgG fraction of immune sera)). Subsequently, or alternatively, the particular antigen or epitope of that antigen that is the desired immunoglobulin target can be immobilized on a column and the immunospecific antibody purified by immunoaffinity chromatography. Purification of immunoglobulins is described, for example, in D.
Wilkinson (The Scientist (The Sciences
t, Inc. , Philadelphia PA), Vol. 14, No. 8 (April 17, 2000), pages 25-28).

Monoclonal Antibody The term “monoclonal antibody” (MAb) or “monoclonal antibody composition” as used herein refers to a unique light chain gene product and a unique heavy chain gene product. It refers to a population of antibody molecules including antibody molecules of a molecular species. In particular, the complementarity determining regions (CDRs) of monoclonal antibodies are identical to all molecules in the population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for the antigen.

Monoclonal antibodies can be prepared using hybridoma methods (eg, Kohler and M).
ilstein, Nature, 256: 495 (1975)). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to produce lymphocytes that produce, or are capable of producing, antibodies that specifically bind the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

The immunizing agent will typically include a protein antigen, fragment thereof or fusion protein thereof. Generally, one of the following: peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. used. The lymphocytes are then fused to an immortalized cell line using suitable fusion factors (eg, polyethylene glycol) to form hybridoma cells (Goding, Monoclo).
nal Antibodies: Principles and Practi
Ce, Academic Press (1986) pp. 59-103). Immortalized cell lines are usually transformed into mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually rat or mouse myeloma cell lines are used. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cell is an enzyme, hypoxanthine guanine phosphoribosyl transferase (H
In the absence of GPRT or HPRT), the culture medium for hybridomas typically contains hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances inhibit the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, which support stable high level expression of antibody by the selected antibody-producing cells and are sensitive to a medium such as HAT medium. Is. More preferred immortalized cell lines are mouse myeloma cell lines, which can be derived from, for example, the Salk Institute Cell Dist.
libration Center, San Diego, California
And American Type Culture Collection,
It can be obtained from Manassas, Virginia. Human myeloma and mouse-human myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (198).
4); Brodeur et al., Monoclonal Antibody Prod.
action Technologies and Applications, M
arcel Dekker, Inc. , New York, (1987) No. 51.
-Page 63).

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

After the desired hybridoma cells have been identified, clones can be subcloned by limiting the dilution procedure and expanded by standard methods.
For example, suitable culture media for this purpose include Dulbecco's M
modified Eagle's Medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in vivo as ascites fluid in mammals.

Monoclonal antibodies secreted by the subclones are isolated from the culture medium or ascites by conventional immunoglobulin purification procedures such as protein A sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. , Can be isolated or purified.

Monoclonal antibodies can also be prepared by recombinant DNA methods (eg, US Pat. No. 4,811).
Method described in No. 6,567). The DNA encoding the monoclonal antibody of the invention is readily prepared using conventional procedures (eg, by using an oligonucleotide probe capable of specifically binding to the genes encoding the heavy and light chains of the mouse antibody). Can be isolated and sequenced. The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which they then
Host cells (eg, monkey C that otherwise does not produce immunoglobulin proteins)
OS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells) to obtain monoclonal antibody synthesis in recombinant host cells. DNA can also be substituted, for example, in the coding sequences for human heavy and light chain constant domains in place of homologous mouse sequences (US Pat. No. 4,816).
, 567; Morrison, Nature 368, 812-13 (199).
4)) or by covalent linkage of all or part of the coding sequence for the non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides may be substituted with the constant domain of the antibody of the invention or with the variable domain of one of the antigen binding sites of the antibody of the invention to produce a chimeric bivalent antibody. obtain.

(Humanized Antibody) The antibody against the protein antigen of the present invention may further include a humanized antibody or a human antibody. These antibodies are suitable for administration to humans who do not elicit an immune response by the human against the administered immunoglobulins. Humanized forms of antibodies include chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, F
ab, Fab ′, F (ab ′) ₂ or another antigen-binding subsequence of an antibody),
They are composed primarily of human immunoglobulin sequences and contain minimal sequences derived from non-human immunoglobulin. Humanization is performed by the method of Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986) by substitution of rodent CDRs or CDR sequences for the corresponding sequences of human antibodies.
); Riechmann et al., Nature, 332: 323-327 (1988).
); Verhoeyen et al., Science, 239: 1534-1536 (1
988)). (See also US Pat. No. 5,225,539.) In some cases, the Fv backbone of human immunoglobulins (fra)
mework) residues are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or scaffold sequences. Generally, a humanized antibody substantially comprises at least one, and typically all two, variable domains. Within these areas,
All or substantially all CDR regions correspond to regions of non-human immunoglobulin and all or substantially all scaffold regions correspond to regions of human immunoglobulin consensus sequences. Humanized antibodies also include immunoglobulin constant regions (
Fc), typically including at least part of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Pres).
ta, Curr. Op. Struct. Biol. , 2: 593-596 (19
92)).

Human Antibodies Fully human antibodies relate to antibody molecules in which the entire sequence of both the light and heavy chains, including the CDRs, arise essentially from human genes. Such an antibody is referred to as “human antibody”,
Or referred to herein as "sufficient human antibody". Human monoclonal antibodies can be prepared by trioma technology; human B cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4:72) and EBV hybridoma technology (Cole et al.) To produce human monoclonal antibodies. , 1985: MONOCLONAL ANTIBODIE
S AND CANCER THERAPY, Alan R.S. Liss, Inc
． , Pp. 77-96). Human monoclonal antibodies can be used in the practice of the present invention, and by the use of human hybridomas (Cote et al., 1983. Proc Natl Acad Sci USA 80: 2026.
-2030) or by transformation of human B cells with Epstein-Barr virus in vitro (Cole et al., 1985: MONO).
CLONAL ANTIBODIES AND CANCER THERAPY
Alan R .; Liss, Inc. , Pp. 77-96)).

In addition, human antibodies have also been described using additional techniques (phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227.
: 381 (1991); Marks et al., J. Am. Mol. Biol. , 222: 58
1 (1991))). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, such as mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Human antibody production was observed upon challenge, which in all respects, including gene rearrangement, assembly, and antibody repertoire, closely resembles that observed in humans. This approach is described, for example, in US Pat. Nos. 5,545,807; 5,545,806;
No. 5,569,825; No. 5,625,126; No. 5,633,42.
5, No. 5,661,016, and Marks et al. (Bio / Techno).
logy 10, 779-783 (1992)); Lonberg et al. (Natur).
re 368 856-859 (1994)); Morrison (Natur.
e 368, 812-13 (1994)); Fishwild et al. (Nature).
Biotechnology 14, 845-51 (1996)); Neub.
erger (Nature Biotechnology 14, 826 (19
96)); and Lonberg and Huszar (Intern. Rev.
． Immunol. 13 65-93 (1995)).

Human antibodies can be further produced using transgenic non-human animals that have been modified to produce more human antibody than the animal's endogenous antibodies in response to challenge with an antigen. (See PCT Publication WO 94/02602.) Endogenous genes encoding immunoglobulin heavy and light chains in non-human hosts are non-functional and human heavy and human light chain immunity. The active locus encoding the globulin is inserted into the genome of the host. For example, human genes are integrated using yeast artificial chromosomes containing essential human DNA segments. Animals that provide all the desired modifications are then obtained as progeny by crossing intermediate transgenic animals that contain less than the full complement of modifications. A preferred embodiment of such non-human animals is the mouse, PCT publications WO 96/33735 and WO 96/3409.
It is referred to as Xenomouse ^™ as disclosed in US Pat. This animal produces B cells that secrete sufficient human immunoglobulin. From animals after immunization with the immunogen of interest (eg, preparation of polyclonal antibodies) or from immortalized B cells (eg, hybridomas producing monoclonal antibodies) of animal origin,
It is possible to obtain the antibody directly. In addition, the gene encoding the immunoglobulin having human variable regions can be recovered and expressed to directly obtain the antibody, or to obtain an analog of the antibody (eg, a single chain Fv molecule, etc.) It can be modified.

An example of a method for making a non-human host lacking expression of an endogenous immunoglobulin heavy chain (exemplified as a mouse) is disclosed in US Pat. No. 5,939,598. It can be obtained by a method comprising the following steps: at least in embryonic stem cells to prevent rearrangement of the locus and to prevent the formation of rearranged immunoglobulin heavy chain locus transcripts. Deleting the J segment gene from one endogenous heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and transgenic mice (somatic and germ cells thereof) Includes a gene encoding a selectable marker) from embryonic stem cells.

Methods of producing an antibody of interest (eg, human antibody) are described in US Pat. No. 5,916,16.
No. 771. The method includes the following steps: introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, an expression vector containing a nucleotide sequence encoding a light chain, Introducing into the other mammalian host cell and fusing these two cells to form a hybrid cell. Hybrid cells express an antibody that includes a heavy chain and a light chain.

In a further refinement of this procedure, methods for identifying clinically relevant epitopes on immunogens, and correlating methods for selecting antibodies that immunospecifically bind to relevant epitopes with high affinity are described in PCT. It is disclosed in publication WO 99/53049.

[0194] According to (F _ab fragments and single chain antibodies) present invention, techniques can be adapted to produce single chain antibodies specific to an antigenic protein of the present invention (e.g., U.S. Patent No. 4,946, (See No. 778)
. In addition, the method can be applied to the construction of _Fab expression libraries (eg, Hu.
Se et al., 1989 Science 246: 1275-1281), allows rapid and effective identification of monoclonal _Fab fragments with the desired specificity for the protein or its derivatives, fragments, analogs or homologs. Antibody fragments containing idiotypes to protein antigens can be produced by techniques known in the art, including (i) F _{(ab ') 2} fragments produced by pepsin digestion of antibody molecules; (ii) _{F (ab ')} F _ab fragments produced by reducing the disulfide bridges of the ₂ fragments; (iii) _{F ab} fragments produced by treatment of the antibody molecule with pepsin and a reducing agent, and (iv
) F _v fragments, but are not limited thereto.

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

Methods of making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello). , Nature, 305: 537-539 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produced a potential mixture of 10 different antibody molecules, of which only one was the correct bispecific. Have a structure. Purification of this precise molecule is usually accomplished by affinity chromatography steps. A similar procedure is described in WO93 / 08829, published May 13, 1993, and Traunec.
ker et al., 1991 EMBO J. et al. 10: 3655-3659.

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion is preferably with an immunoglobulin heavy chain constant domain, including at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. The immunoglobulin heavy chain fusions, and, if desired, the DNA encoding the immunoglobulin light chains, are inserted into separate expression vectors and cotransfected into a suitable host organism. Further details of making bispecific antibodies can be found in, for example, Suresh et al., Methods in Enzymology, 121.
: 210 (1986).

According to another approach described in WO96 / 27011, the interface between pairs of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. The preferred interface is CH3 of the antibody constant domain.
At least a part of the area is included. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). Compensatory "cavities" of identical or similar size for the large side chains are created on the interface of a second antibody molecule by replacing large amino acid side chains with small amino acid side chains (eg alanine or threonine). Is generated. This is more important than other unwanted end products such as homodimers.
A mechanism is provided for increasing the yield of heterodimers.

[0199]   Bispecific antibodies are full-length antibodies or antibody fragments (eg, F (ab ')). _Two Bispecific antibody). Bispecific antibodies from antibody fragments
Techniques for making are described in the literature. For example, a bispecific antibody
, Can be prepared using chemical coupling. Brennan et al., Science 22
9:81 (1985), the intact antibody is proteolytically cleaved to produce F (
ab ')_TwoThe procedure for generating fragments is described. These fragments are
To stabilize vicinal dithiols and prevent intermolecular disulfide formation
In the presence of the dithiol complexing agent sodium arsenite. Then this raw
The Fab 'fragment generated was transformed into a thionitrobenzoate (TBN) derivative.
To be converted. Then one of the Fab'-TNB derivatives is mercaptoethylamido.
It is reconverted to Fab'-thiol by reduction with benzene and this is an equimolar amount of
Mixed with other Fab'-TNB derivatives to form bispecific antibodies. Is generated
Bispecific antibodies can be used as agents for the selective immobilization of enzymes.

In addition, Fab ′ fragments were isolated from E. E. coli and can be chemically coupled to form bispecific antibodies. Shalaby et al., J
． Exp. Med. 175: 217-225 (1992) describe the generation of fully humanized bispecific antibody F (ab ') ₂ molecules. Each Fab 'fragment was isolated from E. are separately secreted from E. coli and subjected to directed chemical coupling in vitro to form bispecific antibodies. Thus, this bispecific antibody that was formed was found to bind cells that overexpress the ErbB2 receptor and normal human T
It can bind cells and trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies are produced using leucine zippers. Kostelny et al. Immuno
l. 148 (5): 1547-1553 (1992). Leucine zipper peptides derived from the Fos and Jun proteins are linked to the Fab ′ portion of two different antibodies by gene fusion. The antibody homodimer is reduced at the hinge region to form a monomer and then reoxidized to form the antibody heterodimer. This method can also be used for the production of antibody homodimers. Holling
er et al., Proc. Natl. Acad. Sci. USA 90: 6444-6.
This "diabody" technique described in 448 (1993) provided an alternative mechanism for making bispecific antibody fragments.
This fragment contains the heavy chain variable region (V _H ) connected to the light chain variable region (V _L ) by a linker that is too short to pair between two domains on the same chain. Therefore, the V _H and V _L domains of one fragment are forced to pair with the complementary V _L and V _H domains of another fragment, which results in 2
One antigen binding site is formed. Another strategy for making bispecific antibody fragments by the use of single chain Fv (sFv) dimers has also been reported. Gruber et al. Immunol. 152: 5368 (1994).

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

Exemplary bispecific antibodies may bind to two different epitopes, at least one of which has its origin in a protein antigen of the invention. Alternatively, the anti-antigenic arm of the immunoglobulin molecule is a T cell receptor molecule (eg, CD2, CD
3, CD28, or B7), or Fc receptor for IgG (Fc
R) (eg, Fc RI (CD64), Fc RII (CD32) and Fc
It can be attached to an arm that binds a trigger molecule on leukocytes such as RIII (CD16)) to focus on the defense mechanism of the cell against cells expressing a particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells that express particular antigens. These antibodies include antigen-binding arms and cytotoxic agents or radionuclide chelators (eg, EOTUBE, DPTA).
, DOTA, or TETA). Another bispecific antibody of interest binds to the protein antigens described herein and also to tissue factor (TF).

Heterozygous Antibodies Heterozygous antibodies are also within the scope of the invention. Heterozygous antibodies are composed of two covalently joined antibodies. Such antibodies are, for example, for targeting immune system cells to unwanted cells (US Pat. No. 4,676,980) and for treatment of HIV infection (WO91 / 00360; WO92 / 200373). ;
EP 03089) has been proposed. It is contemplated that the antibody can be prepared in vitro using methods known in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed by using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate,
And the reagents disclosed in, for example, US Pat. No. 4,676,980.

Manipulation of Effector Function It may be desirable to modify the antibody of the invention with respect to effector function, such as to increase the efficacy of the antibody in treating cancer. For example, cysteine residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. Thus, the homodimeric antibody produced may have improved internalization potential and / or increased complement-mediated cell killing, as well as antibody-dependent cellular cytotoxicity (ADCC). Caron et al.
Exp. Med. 176: 1191-1195 (1192) and Shope.
S.J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with increased antitumor activity have also been described by Wolff et al., Ca.
ncer Research, 53: 2560-2656 (1993), and may be prepared using a heterobifunctional crosslinker. Alternatively, antibodies with dual Fc regions can be engineered, and thereby increased complement lysis and ADC
It may have C possibilities. Stevenson et al., Anti-Cancer Dr
ug Design, 3: 219-230 (1989).

Immunoconjugates The present invention also relates to cytotoxic agents (eg chemotherapeutic agents, toxins (eg enzymatically active toxins of bacterial, fungal, plant or animal origin, Alternatively, fragments thereof), or immunoconjugates comprising an antibody bound to a radioisotope (ie, a radioconjugate).

[0207]   Chemotherapeutic agents useful in making such immunoconjugates are described above.
. Enzymatically active toxins and their fragments that can be used include di-
Phtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (Pse
udomonas aeruginosa), ricin A chain, abrin A chain,
Modecin A chain, α-sarcin, Al
eurites fordii protein, dianthin
Protein, Phytolacca americana protein (PAPI, P
APII, and PAP-S), momordica
  charantia inhibitor, curcin, crotin (
crotin), sapaonaria officinalis (sapona aria off
icinalis) inhibitor, gelonin, mitogillin (
mitogellin, restrictocin, fu
Enomycin (phenomycin), enomycin (enomycin),
And trichothecene. Various radioactivity
Nuclides are available for the production of radioactively bound antibodies. For example, ²¹² Bi,¹³¹I,¹³¹In,⁹⁰Y, and¹⁸⁶Re is mentioned.

Conjugates of antibodies and cytotoxic agents can be conjugated to various bifunctional protein binding agents (
For example, N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), a bifunctional derivative of an imide ester (eg, dimethyl adipimidate HCL),
Active esters (eg, disuccinimidyl suberate), aldehydes (eg, glutaraldehyde), bisazide compounds (eg, bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (eg, bis- ( p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (eg triene 2,6-diisocyanate), and bis-active fluorine compounds (eg 1,5-difluoro-2,4-dinitrobenzene)). It For example, the ricin immunotoxin is Vitetta.
Et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for coupling radionuclides to antibodies. See WO94 / 11026.

In another embodiment, for use in tumor pre-targeting, the antibody is
A "receptor" (eg, streptavidin) may be bound, where the antibody-receptor conjugate is administered to a patient, followed by removal of unbound conjugate from the circulation using a clearing agent, followed by cytotoxicity. "Ligands" that bind to sex drugs in turn (
For example, avidin) is administered.

(NOVX Recombinant Expression Vector and Host Cell) Another aspect of the present invention relates to a vector, preferably an expression vector, containing a nucleic acid encoding the NOVX protein, or a derivative, fragment, analog or homolog thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been attached. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of spontaneous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors, such as non-episomal mammalian vectors, integrate into the host cell's genome when introduced into the host cell, and thereby replicate in synchronization with the host's genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such a vector is referred to herein as an "expression vector". In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In the present specification, "plasmid" and "vector" refer to
Since this plasmid is the most commonly used form of vector, it can be used interchangeably. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. .

The recombinant expression vector of the invention comprises a nucleic acid of the invention in a form suitable for the expression of that nucleic acid in a host cell, which is the host cell in which the recombinant expression vector is used for expression. It is meant to include one or more regulatory sequences selected on the basis of which are operably linked to the expressed nucleic acid sequence. In a recombinant expression vector, "operably linked" allows the nucleotide sequence of interest to allow the expression of that nucleotide sequence (eg, in an in vitro transcription / translation system or when the vector is introduced into a host cell, the host (In the cell) is intended to be linked to regulatory sequences in such a manner.

The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, by Goeddel, GENE EXPRESSION TEC.
HNOLOGY: METHODS IN ENZYMOLOGY 185, Ac
acoustic Press, San Diego, Calif. (1990). Regulatory sequences include sequences that direct constitutive expression of nucleotide sequences in many types of host cells, and sequences that direct expression of nucleotide sequences only in particular host cells (eg, tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of transformed host cell, the level of expression of the desired protein and the like. The expression vector of the present invention is introduced into a host cell, whereby a protein or peptide (including fusion protein or fusion peptide) encoded by a nucleic acid as described herein (eg, NOVX protein, NOVX protein). Mutated forms, fusion proteins, etc.) can be produced.

The recombinant expression vector of the invention may be used in prokaryotic or eukaryotic cells,
It can be designed for the expression of NOVX proteins. For example, the NOVX protein can be expressed in bacterial cells (eg Escherichia coli), insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are Goeddel, GENE EXPRESSI
ON TECHNOLOGY: METHODS IN ENZYMOLOGY
185, Academic Press, San Diego, Calif. (
1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. . Fusion vectors add a number of amino acids to the protein encoded therein, usually at the amino terminus of recombinant proteins. Such fusion vectors typically serve three purposes: (i) increasing the expression of the recombinant protein; (ii) increasing the solubility of the recombinant protein; and ( iii) To assist in the purification of recombinant proteins by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety, and after purification of the fusion protein, the recombinant protein allows the recombinant protein to be separated from the fusion moiety. To Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase. As a typical fusion expression vector, pGEX (Pharmacia Biotech Inc) that fuses glutathione S-transferase (GST), maltose E-binding protein, or protein A to a target recombinant protein;
Smith and Johnson 1988. Gene 67: 31-40
), PMAL (New England Biolabs, Beverly, M.
ass. ) And pRIT5 (Pharmacia, Piscataway, N.
． J. ) Is mentioned.

Appropriate inducible unfused E. An example of an E. coli expression vector is pTrc (Am
runn et al. (1988) Gene 69: 301-315) and pET 11
d (Studier et al., GENE EXPRESSION TECHNOLOG
Y: METHODS IN ENZYMOLOGY 185, Academic
Press, San Diego, Calif. (1990) 60-89).

E. One strategy for maximizing recombinant protein expression in E. coli is to express the protein in a host bacterium with impaired ability to proteolytically cleave the recombinant protein. For example, Gottesman,
GENE EXPRESSION TECHNOLOGY: METHODS I
N ENZYMOLOGY 185, Academic Press, San
Diego, Calif. (1990) 119-128. Another strategy is that the individual codons for each amino acid are to modify the nucleic acid sequence of the nucleic acid inserted into the expression vector so that it is preferentially utilized in E. coli (eg Wada et al., 1992. Nucl. Acids.
Res. 20: 2111-2118). Such alterations of the nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[0217] In another embodiment, the NOVX expression vector is a yeast expression vector.
Examples of vectors for expression in the yeast Saccharomyces cerevisiae include pYepSec1 (Baldari et al., (1987) EMBO.
J 6: 229-234), pMFa (Kurjan and Herskowi.
tz, 1982. Cell 30: 933-943), pJRY88 (Schu
ltz et al., 1987. Gene 54: 113-123), pYES2 (Inv.
itrogen Corporation, San Diego, Calif.
), And picZ (InVitrogen Corp., San Diego.
, Calif. ) Is mentioned.

Alternatively, NOVX can be expressed in insect cells using a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (eg, SF9 cells) include pAc series (
Smith et al., 1983. Mol. Cell. Biol. 3: 2156-216
5) and the pVL series (Lucklow and Summers, 1989.
Virology 170: 31-39).

In yet another embodiment, the nucleic acids of the invention are expressed in mammalian cells using mammalian expression vectors. An example of a mammalian expression vector is pCDM8
(Seed, 1987. Nature 329: 840) and pMT2PC (
Kaufman et al., 1987. EMBO J.M. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often
Provided by the regulatory elements of the virus. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, eg, Sambrook et al., MOLECUL.
AR CLONING: A LABORATORY MANUAL. Second edition, C
old Spring Harbor Laboratory, Cold Sp
ring Harbor Laboratory Press, Cold Sp
ring Harbor, N.M. Y. , 1989, chapters 16 and 17.

In another embodiment, the recombinant mammalian expression vector is capable of directing the expression of the nucleic acid preferentially in a particular cell type (eg, expressing the nucleic acid using tissue-specific regulatory elements). .. Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (
Liver-specific; Pinkert et al., 1987. Genes Dev. 1: 268-
277), lymphoid-specific promoter (Calame and Eaton, 198).
8. Adv. Immunol. 43: 235-275), especially the T cell receptor promoter (Winoto and Baltimore, 1989. EMBO.
J. 8: 729-733) and immunoglobulins (Banerji et al., 198).
3. Cell 33: 729-740; Queen and Baltimore,
1983. Cell 33: 741-748), neuron-specific promoters (eg, neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA).
86: 5473-5477), pancreas specific promoter (Edlund et al., 19).
85. Science 230: 912-916), and mammary gland-specific promoters (eg, milk whey promoter; US Pat. No. 4,873,316 and EP 264,166). Developmentally regulated promoters are also included (eg, the murine hox promoter (Kessel and Gruss, 1990. Science 249.
: 374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
.

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in the antisense orientation. That is, the DNA molecule expresses an RNA molecule that is antisense to NOVX mRNA (DN
(By transcription of the A molecule) is operably linked to the regulatory sequences in a manner that enables
A regulatory sequence operably linked to the nucleic acid cloned in the antisense orientation can be chosen which directs the continuous expression of the antisense RNA molecule in various cell types. For example, viral promoters and / or enhancers, or regulatory sequences that direct constitutive, tissue-specific, or cell-type specific expression of antisense RNA may be selected. The antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus, where the antisense nucleic acid is produced under the control of a high efficiency regulatory region, the activity of which is introduced into the vector. Cell type. For a discussion of the regulation of gene expression using antisense genes, see, eg, Weintraub et al., “Ant.
Sense RNA as a molecular tool for g
Genetic analysis ”, Reviews-Trends in G
See enetics, Volume 1 (1) 1986.

Another aspect of the present invention relates to a host cell into which the recombinant expression vector of the present invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell, but to the progeny or potential progeny of such a cell. Such progeny may, in fact, not be identical to the parental cell, as the particular modification may be present in the next generation, either due to mutations or environmental influences, but is still referred to herein. Within the scope of the term as used in.

The host cell can be any prokaryotic or eukaryotic cell. For example, N
The OVX protein is a bacterial cell (eg, E. coli), insect cell, yeast or mammalian cell (eg, human cell, Chinese hamster ovary cell (CHO).
) Or COS cells). Other suitable host cells are known to those of skill in the art.

Vector DNA may be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to various art-recognized techniques for introducing exogenous nucleic acid (eg, DNA) into a host cell. Are intended to include and include calcium phosphate or calcium chloride co-precipitation, DEAE dextran mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells are described by Sambrook et al. (MOLECULAR CL
ONING: A LABORATORY MANUAL. Second Edition, Cold S
pring Harbor Laboratory, Cold Spring
Harbor Laboratory Press, Cold Spring
Harbor, N.M. Y. , 1989) and other laboratory manuals.

For stable transfection of mammalian cells, it is known that only a fraction of the cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. .. To identify and select for these elements, a gene encoding a selectable marker (eg, resistance to antibiotics) is generally introduced into the host cell along with the gene of interest. Various selectable markers include markers that confer resistance to drugs (eg, G418
, Hygromycin, and methotrexate). The nucleic acid encoding the selectable marker can be introduced into the host cell on the same vector as the vector encoding NOVX or can be introduced on a separate vector. Cells that are stably transfected with the introduced nucleic acid can be identified by drug selection (
For example, cells that have incorporated the selectable marker gene will survive, while others will die).

Host cells of the invention (eg, prokaryotic and eukaryotic host cells in culture) can be used to produce (ie, express) NOVX protein. Accordingly, the invention further provides methods for producing NOVX proteins using the host cells of the invention. In one embodiment, the method is NO
Culturing the host cell of the present invention (where the recombinant expression vector encoding the NOVX protein has been introduced) in a suitable medium in which the VX protein is produced. In another embodiment, the method further comprises the step of isolating NOVX from the medium or host cells.

Transgenic NOVX Animals The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, the host cell of the invention is NOVX.
A fertilized oocyte or embryonic stem cell into which a protein coding sequence has been introduced. Such host cells can then be used to produce non-human transgenic animals, where the exogenous NOVX sequences are altered by their genomic or homologous recombinant animals (where the endogenous NOVX sequences are altered. Has been introduced). Such animals are used to study the function and / or activity of NOVX proteins,
And NOVX proteins are useful for identifying and / or assessing modulators of activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, wherein these animals are One or more cells of which contain the transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians and the like. A transgene is exogenous DNA that integrates into the genome of a cell (from which cells a transgenic animal develops) and remains in the genome of a mature animal, thereby producing one or more cells of this transgenic animal. Directs expression of the encoded gene product in the form or tissue. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, wherein the endogenous NOVX gene is the endogenous NOVX gene. It has been modified by homologous recombination between a gene and an exogenous DNA molecule introduced into an animal cell (eg, an animal embryonic cell) prior to animal development.

The transgenic animals of the present invention may be prepared by introducing a nucleic acid encoding NOVX (by introducing into the male pronucleus of a fertilized oocyte (eg, by microinjection, retroviral infection)) and by this oocyte. Can be generated in a pseudopregnant female foster animal SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,
The sequence containing 19 or 21 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue of the human NOVX gene (eg mouse NOV
X gene) can be isolated based on hybridization to human NOVX cDNA (further described above) and used as a transgene. Intron sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. Tissue-specific regulatory sequence (s) is operably linked to the NOVX transgene to direct expression of the NOVX protein to particular cells. Methods for producing transgenic animals, especially animals such as mice, via embryo manipulation and microinjection are conventional in the art and are described, for example, in US Pat. No. 4,736,866. No. 4,870,009; and No. 4,873.
, 191; and Hogan 1986, MANIPULATING TH.
E MOUSE EMBRYO, Cold Spring Harbor La
laboratory Press, Cold Spring Harbor, N.M.
Y. It is described in. Similar methods are used for the production of other transgenic animals. Transgenic founders have NOVX in their genome
Presence of transgene and / or NOVX mR in tissues or cells of the animal
It can be identified based on the expression of NA. Then the transgenic primary animal
It can be used to breed additional animals carrying the transgene. Furthermore, N
Transgenic animals carrying a transgene encoding an OVX protein are:
In addition, it can be bred to other transgenic animals with other transgenes.

Deletions, additions, or substitutions have been introduced to produce a homologous recombinant animal, thereby altering (eg, functionally disrupting) the NOVX gene, at least a portion of the NOVX gene. A vector containing is prepared. The NOVX gene is a human gene (eg, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 1).
9 or 21 DNA), but more preferably a non-human homologue of the human NOVX gene. For example, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 1
Mouse homologs of 5, 17, 19 or 21 human NOVX genes can be used to construct homologous recombination vectors suitable for altering the endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that upon homologous recombination, the endogenous NOVX gene is functionally disrupted (ie, no longer encodes a functional protein; also referred to as a "knockout" vector). .

Alternatively, the vector is designed such that upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered, but still encodes a functional protein (eg, , Upstream regulatory regions may be altered, thereby altering expression of the endogenous NOVX protein). In the homologous recombination vector,
The altered portion of the NOVX gene is modified by the additional nucleic acid of the NOVX gene,
Flanked by its 5'and 3'ends, it enables homologous recombination to occur between the exogenous NOVX gene carried by the vector and the endogenous NOVX gene in embryonic stem cells. The additional flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of contiguous DNA (
Both the 5'end and the 3'end) are included in the vector. For example, regarding the description of homologous recombination vectors, Thomas et al. (1987) Cell 51: 503.
checking ... The vector is then introduced into an embryonic stem cell line (eg, by electroporation) and the introduced NOVX gene is transferred to endogenous NO.
Cells that have undergone homologous recombination with the VX gene are selected (eg, Li et al. (1992).
), Cell 69: 915).

The selected cells are then injected into the blastocyst of an animal (eg mouse) to form an aggregated chimera. For example, TERATO from Bradley (1987)
CARINOMAS AND EMBRYONIC STEM CELLS:
A PRACTICAL APPROACH, edited by Robertson, IRL,
See Oxford pages 113-152. The chimeric embryo can then be transferred to a suitable pseudopregnant female foster animal, and the embryo is allowed to rest for a period of time. Offspring carrying homologous recombination DNA in their germ cells may be used to breed animals,
Here, all cells of this animal contain this homologous recombinant DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further below; Bradley (1991) Cur.
r. Opin. Biotechnol. 2: 823-829; PCT International Publication Numbers: WO90 / 11354; WO91 / 01140; WO92 / 0968; and WO93 / 04169.

In another embodiment, non-human transgenic animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre / loxP recombinase system of bacteriophage P1.
For a description of the cre / loxP recombinase system, see, eg, Lakso et al.
1992, Proc. Natl. Acad. Sci. USA 89: 6322 ~
See 6236. Another example of a recombinase system is Saccharomyc.
es cerevisiae FLP recombinase system (O'Gorma
n, et al., 1991, Science 251: 1351-1355).
. If the cre / loxP recombinase system is used to regulate transgene expression, then an animal containing the transgene encoding both the Cre recombinase and the selected protein is required. Such animals can be "double-stranded", for example, by crossing two transgenic animals, one containing the transgene encoding the selected protein and the other containing the transgene encoding the recombinase. '' Can be provided by the construction of transgenic animals.

The transgenic non-human animal clones described herein also contain W
ilmut et al., 1997, Nature 385: 810-813. Briefly, cells from transgenic animals (
For example, somatic cells) can be isolated and induced, exit the growth cycle and enter G ₀ phase. The quiescent cells can then be fused, eg, by use of electric pulses, to isolated oocytes from the same animal from which the quiescent cells are isolated. The reconstructed oocytes are then cultured, which allows them to develop into morula or undifferentiated germ cells and then transferred to pseudopregnant female foster animals. The progeny of the female Foster animal is a clone of the animal from which the cells (eg, somatic cells) are isolated.

Pharmaceutical Compositions NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies of the invention (also referred to herein as “active compounds”), and derivatives, fragments, analogs thereof, And homologues can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include a nucleic acid molecule, a protein or antibody, and a pharmaceutically acceptable carrier. As used herein,
"Pharmaceutically acceptable carrier" includes any and all solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic and delaying agents, etc., which are suitable for pharmaceutical administration. Is intended to include. A suitable carrier is Remington's P, a standard reference text in the field.
described in the latest edition of the Pharmaceutical Sciences, which is hereby incorporated by reference. Preferred examples of such carriers or excipients include water, saline, finger solution, dextrose solution, and 5%
Includes, but is not limited to, human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except to the extent of any conventional vehicle or drug that is incompatible with the active compound, its use in the composition is intended. Supplementary active compounds can also be incorporated into the compositions.

The antibodies disclosed herein can be formulated as immunoliposomes. Liposomes containing this antibody are prepared by methods known in the art (eg, E
pstein et al., Proc. Natl. Acad. Sci. USA, 82:36.
88 (1985); Hwang et al., Proc. Natl. Acad. Sci. U
SA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545). Liposomes with enhanced circulation time are disclosed in US Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse layer evaporation method with a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size, yielding liposomes with the desired diameter. Fab 'fragments of the antibodies of the present invention are described by Martin et al. Biol. C
hem. , 257: 286-288 (1982), and can be conjugated to liposomes via a disulfide exchange reaction. Chemotherapeutic agents (eg Doxo)
rubicin) may be optionally contained within the liposome. Gabizon
Et al., J. National Cancer Inst. , 81 (19): 148
4 (1989).

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (eg, intravenous, intradermal, subcutaneous), oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following ingredients: sterile vehicle (water for injection, saline solution, fixed oils, Polyethylene glycol, glycerin, propylene glycol or other synthetic solvents); antibacterial agents (eg benzyl alcohol or methylparaben); antioxidants (eg ascorbic acid or sodium bisulfite)
Chelating agents (eg ethylenediaminetetraacetic acid (EDTA)); buffers (eg acetate, citrate or phosphate); and agents for adjusting tonicity (eg sodium chloride or dextrose). pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include saline, bacteriostatic water, Crem.
Ophor EL ^™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be easy to inject.
It should be fluid to the extent that ty) is present. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example: water, ethanol, polyols such as glycerol, propylene glycol, and liquid polyethylene glycols, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size for dispersion and by the use of surfactants. Prevention of the action of microorganisms involves the treatment of various antibacterial and antifungal agents (eg paraben, chlorobutanol,
Phenol, ascorbic acid, thimerosal, etc.). In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount, eg, a NOVX protein or anti-NOVX antibody, in a suitable solvent with one or a combination of the above-listed components, if necessary. , Followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the method of preparation is vacuum drying and freeze drying, whereby a powder of the active ingredient and any further desired ingredients from that solution which has been previously sterile filtered. To get

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. The oral composition also
It can be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished. Then it is exhaled or swallowed. Pharmaceutically compatible binders, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules,
Lozenges and the like can include any of the following ingredients or compounds having similar properties: Binders such as microcrystalline cellulose, gum tragacanth or gelatin.
Excipients (eg starch or lactose), disintegrants (eg alginic acid), Primogel, or corn starch; lubricants (eg magnesium stearate or Sterotes); glidants (eg colloidal silicon dioxide); A sweetener (eg sucrose or saccharin); or a flavoring agent (eg peppermint, methyl salicylate, or orange flavor).

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

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished by nasal spray or suppositories. For transdermal administration, the active compound may be an ointment, which is generally known in the art,
Formulated in ointment, gel, or cream.

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

In one embodiment, the active compound is prepared with a carrier that protects the compound against rapid elimination from the body (eg, controlled release formulations),
This includes implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
Methods for the preparation of such formulations will be apparent to those of skill in the art. These materials are also commercially available from Alza Corporation and Nova Phar.
scientifics, Inc. Commercially available from Liposomal suspensions, including liposomes targeted to infected cells, containing monoclonal antibodies against viral antigens, can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically individual units suitable as unitary dosages for the subject to be treated; each unit being associated with a required pharmaceutical carrier. And a predetermined amount of active compound calculated to produce the desired therapeutic effect. Details regarding the dosage unit forms of the present invention are given by the unique properties of the active compound, and the particular therapeutic effect achieved, as well as limitations inherent in the art of formulating such active compounds for treatment of individuals. Determined or directly dependent on them.

The nucleic acid molecule of the present invention can be inserted into a vector and used as a gene therapy vector. Gene therapy vectors can be administered to a subject by, for example, intravenous injection, topical administration (see US Pat. No. 5,328,470) or stereotactic injection (eg, Chen et al. (1994) Proc. Natl. Sci. USA). .91: 305
4-3057)). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively,
The complete gene delivery vector can be produced intact from recombinant cells (eg, retroviral vectors) and this pharmaceutical preparation produces a gene delivery system 1.
The above cells may be included.

Antibodies that specifically bind the proteins of the invention, as well as other molecules identified by the screening assays disclosed herein, may be used in the form of pharmaceutical compositions for the treatment of various disorders. Can be administered. Principles and considerations regarding the preparation of such compositions, as well as guidance in selecting the ingredients, can be found in, for example, Remin.
gton's The Science And Practice Of Pha
rmacy 19th edition (Alfonso R. Gennaro et al., Ed.) Mack
Pub. Co. , Easton, Pa: 1995; Drug Absorbt.
Ion Enhancement: Concepts, Posibiliti
es, Limitations, And Trends, Harwood Ac
academic Publishers, Langhorne, Pa. , 1994
; And Peptide And Protein Drug Deliver
y (Advances In Parental Sciences, Vo
l. 4), 1991, M .; Dekker, New York. Provided to. If the antigenic protein is intracellular and the intact antibody is used as an inhibitor, it is preferred to internalize the antibody. However, liposomes can also be used to deliver the antibody or antibody fragment to cells. When antibody fragments are used, the minimal inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules can be designed that maintain the ability to bind a target protein sequence based on the variable region sequences of the antibody. Such peptides may be chemically synthesized and / or recombinant DN
A technology. See, for example, Marasco et al., 1993 Proc.
． Natl. Acad. Sci. USA, 90: 7889-7893. The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably compounds with complementary activities that do not adversely affect each other. Alternatively, or additionally, the composition may include an agent that enhances its function (eg, a cytotoxic agent, cytokine, chemotherapeutic agent or antiproliferative agent). Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The active ingredients are also colloidal drug delivery, for example in microcapsules prepared by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively. It can be trapped in systems such as liposomes, albumin microparticles, microemulsions, nanoparticles, and nanocapsules or macroemulsions.

The formulations used for in vivo administration are sterile. This is easily accomplished by filtration through a sterile filtration membrane.

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped particles, eg films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)).
Polylactide (US Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
Degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT ^™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

The pharmaceutical composition may be included in a container, package, or dispenser together with instructions for administration.

Screening and Detection Methods The isolated nucleic acid molecules of the invention are used to express NOVX proteins (eg, via recombinant expression vectors in host cells during gene therapy applications), NOVX mRNA (eg, in a biological sample) or NO
Genetic lesions in the VX gene can be detected and NOVX activity can be regulated as described further below. In addition, NOVX proteins screen for drugs or compounds that modulate NOVX protein activity or expression, as well as N
It can be used to treat disorders characterized by insufficient or excessive production of OVX protein or reduced production of NOVX protein form or aberrant activity compared to NOVX wild type protein. Furthermore, the anti-NOVX of the present invention
Antibodies can be used to detect and isolate NOVX proteins as well as regulate NOVX activity. For example, NOVX activity includes growth and differentiation, antibody production, and tumor growth.

The invention further relates to novel agents identified by the screening assays described herein, and their use for the treatments described above.

Screening Assays The present invention relates to modulators, ie, candidate compounds or agents that bind to a NOVX protein or have a stimulatory or inhibitory effect on, for example, NOVX protein expression or NOVX protein activity, Alternatively, a method (also referred to herein as a "screening assay") for identifying a test compound or test agent (eg, peptide, peptidomimetic, small molecule or other drug) is provided. The present invention also includes compounds identified in the screening assays described herein.

In one embodiment, the invention provides a candidate for binding to, or modulating the activity of, a membrane-bound form of a protein or polypeptide of NOVX, or a biologically active portion thereof. An assay is provided for screening compounds or test compounds. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, these libraries include: biological libraries; spatial libraries. Parallel solid-phase or solution-phase libraries accessible to the library; synthetic library methods that require deconvolution; "one-bead-one-compound" library methods; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, but the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers or compounds (Lam (1997) Anticancer Drug D).
design 12: 145).

As used herein, “small molecule” is meant to refer to a composition having a molecular weight of less than about 5 kD, most preferably less than about 4 kD. Small molecules can be, for example, nucleic acids, peptides, polypeptides, peptidomimetics, sugars, lipids or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures (eg fungal, bacterial or algae extracts) are known in the art and can be screened using any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can be found in the art, for example: DeWitt et al. (1993) Proc. Natl. Acad.
Sci. U. S. A. 90: 6909; Erb et al. (1994) Proc. Nat
l. Acad. Sci. U. S. A. 91: 11422; Zuckermann
(1994) J. et al. Med. Chem. 37: 2678; Cho et al. (1993).
Science 261: 1303; Carrell et al. (1994) Angew.
． Chem. Int. Ed. Engl. 33: 2059; Carell et al. (19
94) Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop et al. Med. Chem. 37: 1233.

Compound libraries are available in solution (see, eg, Houghten (1992).
Biotechniques 13: 412-421) or on beads (L
am (1991) Nature 354: 82-84) on chip (Fodor)
(1993) Nature 364: 555-556), bacteria (Ladner.
US Pat. No. 5,223,409), spores (Ladner US Pat. No. 5,23)
3,409), plasmid (Cull et al. (1992) Proc. Natl. A.
cad. Sci. USA 89: 1865-1869) or on phage (Sc
Ott and Smith (1990) Science 249: 386-390.
Devlin (1990) Science 249: 404-406; Cwi
rla et al. (1990) Proc. Natl. Acad. Sci. U. S. A. 8
7: 6378-6382; Felici (1991) J. Mol. Biol. Two
22: 30-310; Ladner, U.S. Pat. No. 5,233,409).

[0258] In one embodiment, the assay is a cell-based assay, wherein:
Cells expressing a membrane-bound form of the NOVX protein, or a biologically active portion thereof, on the cell surface are contacted with a test compound, and the test compound is a NOVX protein.
The ability to bind the X protein is determined. For example, the cell can be of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope label or an enzyme label, such that the NOVX protein of the test compound or its biologically active label is bound. Binding to the moiety can be achieved by being able to be determined by detecting its labeled compound in the complex. For example, test ^{compounds, 1 25 I, 35 S,} 14 C or can be labeled either directly or indirectly with ^{3 H,,} and its radioactive isotopes, by direct counting of radioemission or scintillation, It can be detected by counting. Alternatively, the test compound can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determining conversion of the appropriate substrate to product. . In one embodiment, the assay involves contacting cells expressing a membrane-bound form of the NOVX protein, or a biologically active portion thereof, on its cell surface with a known compound that binds NOVX, Forming a mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NOVX protein, wherein the test compound interacts with the NOVX protein. The step of determining potency is such that this test compound has a NOVX
Determining the ability to bind preferentially to the protein or biologically active portion thereof.

[0259] In another embodiment, the assay is a cell-based assay, which comprises:
Contacting a cell expressing a membrane-bound form of the NOVX protein or a biologically active portion thereof on the cell surface with a test compound, as well as the test compound comprising:
The step of determining the ability to modulate (eg, stimulate or inhibit) the activity of the NOVX protein or biologically active portion thereof. This test compound is NOV
Determining the ability to modulate the activity of X or a biologically active portion thereof can be accomplished, for example, by N
This can be accomplished by determining the ability of the OVX protein to bind to or interact with NOVX target molecules. As used herein, a "target molecule" is a molecule with which a NOVX protein naturally binds or interacts, eg, a molecule on the cell surface that expresses a NOVX interacting protein, a second A molecule on the cell surface, a molecule in the extracellular environment, a molecule that associates with the inner surface of the cell membrane, or a cytoplasmic molecule. The NOVX target molecule can be a non-NOVX molecule or a NOVX protein or polypeptide of the invention. In one embodiment, the NOVX target molecule is a component of a signal transduction pathway that involves an extracellular signal through the cell membrane and into the cell (eg, the compound is a membrane-bound NOVX.
It promotes the transmission of signals generated by binding to molecules. Its target is
For example, it may be a second intracellular protein having catalytic activity, or a protein that facilitates the association of downstream signal molecules with NOVX.

Determining the ability of a NOVX protein to bind to or interact with a NOVX target molecule is accomplished by one of the above methods for determining direct binding.
Can be achieved. In one embodiment, determining the ability of a NOVX protein to bind to or interact with a NOVX target molecule can be accomplished by determining the activity of that target molecule. For example, the activity of this target molecule is
Detecting the induction of its target cellular second messenger (ie intracellular Ca ²⁺ , diacylglycerol, IP _3, etc.), detecting the catalytic / enzymatic activity of the target to the appropriate substrate, the reporter gene (detectable Detection of induction of a NOVX responsive regulatory element operably linked to a nucleic acid encoding a different marker (eg, luciferase), or a cellular response (eg, cell viability, cell differentiation, or cell proliferation) Can be determined by detecting

In yet another embodiment, the assay of the present invention is a cell-free assay,
Contacting the OVX protein or biologically active portion thereof with a test compound, as well as determining the ability of the test compound to bind to the NOVX protein or biologically active portion thereof. The binding of this test compound to the NOVX protein can be determined either directly or indirectly, as described above. In one such embodiment, the assay comprises contacting a NOVX protein or biologically active portion thereof with a known compound that binds NOVX to form an assay mixture, the assay mixture being tested. Contacting the compound, as well as determining the ability of the test compound to interact with the NOVX protein, wherein determining the ability of the test compound to interact with the NOVX protein comprises: , Determining the ability to preferentially interact with NOVX, or a biologically active portion thereof, as compared to known compounds.

[0262] In yet another embodiment, the assay is a cell-free assay, NOVX.
Determining the step of contacting the protein or biologically active portion thereof with a test compound, as well as the ability of the test compound to modulate (eg, stimulate or inhibit) the activity of the NOVX protein or biologically active portion thereof. Including steps. Determining the ability of this test compound to modulate the activity of NOVX is accomplished, for example, by determining the ability of the NOVX protein to bind to the NOVX target molecule by one of the methods described above for determining direct binding. Can be done. In an alternative embodiment, the determination of the ability of this test compound to modulate the activity of NOVX protein is NO.
This can be accomplished by determining the ability of the VX protein to further regulate the NOVX target molecule. For example, the catalytic / enzymatic activity of the target molecule on the appropriate substrate can be determined as described above.

In yet another embodiment, the cell-free assay comprises contacting a NOVX protein or biologically active portion thereof with a known compound that binds a NOVX protein to form an assay mixture, the assay mixture Contacting the test compound with a test compound, as well as determining the ability of the test compound to interact with the NOVX protein, wherein determining the ability of the test compound to interact with the NOVX protein comprises: The step of determining the ability to bind preferentially to a NOVX target molecule or to modulate the activity of that target molecule preferentially.

The cell-free assay of the present invention is acceptable for the use of both soluble or membrane bound forms of NOVX proteins. For cell-free assays that include a membrane-bound form of the NOVX protein, it may be desirable to utilize a solubilizer such that the membrane-bound form of the NOVX protein is maintained in solution. Examples of such solubilizers include nonionic surfactants such as n-octyl glucoside, n-dodecyl glucoside, n-dodecyl maltoside, octanoyl-N-methylglucamide, decanoyl-N. -Methylglucamide, Triton®
X-100, Triton (registered trademark) X-114, Thesit (registered trademark)
, Isotridecyl poly (ethylene glycol ether) _n (Isotridec
ypoly (ethylenglycol ether) _n , N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, 3- (3-
Colamidopropyl) dimethylammonio-1-propanesulfonate (3- (3
-Cholamidopropyl) dimethylammniol-1-
Propane sulphonate (CHAPS), or 3- (3-colamidopropyl) dimethylammonio-2-hydroxy-1-propanesulfonate (3- (3-cholamidopropyl) dimethylamine)
iol-2-hydroxy-1-propane sulphonate) (C
HAPSO).

In more than one embodiment of the above assay methods of the invention, either the NOVX protein or its target molecule is immobilized to facilitate separation of the complex form from the uncomplexed form of one or both of the proteins. And applied to the automation of the assay. The binding of the test compound to the NOVX protein, or the interaction of the NOVX protein with the target molecule in the presence and absence of the candidate compound, can be carried out in any vessel suitable to house these reactants. , Can be achieved. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein may be provided that adds a domain that allows one or both of the proteins to bind to the matrix. For example, a GST-NOVX fusion protein or a GST target fusion protein may be glutathione sepharose beads (Sigma).
Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which can then be combined with the test compound, or the test compound and non-adsorbed target protein, or NOV.
Combined with any of the X proteins, and the mixture is incubated under conditions that contribute to complex formation (eg, physiological conditions with respect to salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, in the case of beads the matrix is immobilized and the complex is directly conjugated, eg as described above (supra). Either indirectly or indirectly. Alternatively, the complex can be dissociated from the matrix and the level of NOVX protein binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the NOVX protein, or its target molecule, can be immobilized utilizing the binding of biotin and streptavidin. The biotinylated NOVX protein, or target molecule, can be prepared from biotin-NHS (N-hydroxysuccinimide) using techniques well known in the art (eg, biotinylation kit, Pierce Che.
musicals, Rockford, Ill. ), And in a well of a 96-well plate (Pierce Chemical) coated with streptavidin. Alternatively, the NOVX protein, or an antibody reactive with the target molecule but not interfering with the binding of the NOVX protein to its target molecule, can be derivatized to the wells of the plate and the unbound target or NOVX protein is It can be trapped in the well by the binding of the antibody. Methods for detecting such complexes include, in addition to those described above for GST-immobilized complexes, NO
Immunodetection of the complex using an antibody reactive with the VX protein or target molecule,
And NOVX proteins, or enzyme-linked assays that rely on detection of enzymatic activity with respect to the target molecule.

In another embodiment, modulators of NOVX protein expression are identified in a method of contacting a cell with a candidate compound and determining the expression of NOVX mRNA or protein in the cell. NOVX m in the presence of a candidate compound
RNA or protein expression levels are shown to be NOVX in the absence of candidate compounds.
The mRNA or protein expression level is compared. The candidate compound is then
Based on this comparison, it can be identified as a modulator of NOVX mRNA or protein expression. For example, if the expression of NOVX mRNA or protein is greater (ie, statistically significantly greater) in the presence of the candidate compound than in the absence thereof, the candidate compound is a stimulator of NOVX mRNA or protein expression. Identified as. Alternatively, if NOVX mRNA or protein expression is less (statistically significantly less) in the presence of the candidate compound than in the absence thereof, then the candidate compound is identified as an inhibitor of NOVX mRNA or protein expression. . The expression level of NOVX mRNA or protein in the cell can be determined by the methods described herein for detecting NOVX mRNA or protein.

In yet another aspect of the invention, NOVX proteins are used as “bait proteins” in two-hybrid or three-hybrid assays (eg, US Pat. No. 5,283,317; Zervos et al. (19).
93) Cell 72: 223-232; Madura et al., 1993 J. Am. Bi
ol. Chem. 268: 12046-12054; Bartel et al., 1993.
Biotechniques 14: 920-924; Iwabuchi et al.,
1993 Oncogene 8: 1693-1696; and Brent W.
O94 / 10300), binds to or interacts with NOVX (“NOVX binding protein” or “NOVX-bp”), and NO
Other proteins that regulate VX activity can be identified. Such NOVX binding proteins can also be used as, for example, NOVX as upstream or downstream elements of the NOVX pathway.
It appears to be involved in signal transduction by the X protein.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, this assay utilizes two different DNA constructs. In one construct, the gene encoding NOVX is DN of a known transcription factor (eg, GAL-4).
It is fused to the gene encoding the A-binding domain. In the other construct, D
A DNA sequence encoding an unidentified protein ("prey" or "sample") from a library of NA sequences is fused to a gene encoding the activation domain of a known transcription factor. The DNA binding and activation domains of this transcription factor are in close proximity when the "bait" and "prey" proteins can interact in vivo to form a NOVX-dependent complex. By virtue of its proximity, a reporter gene operably linked to a transcriptional regulatory site responsive to the transcription factor (
For example, it allows transcription of LacZ). Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain a cloned gene encoding a protein that interacts with NOVX.

The present invention further relates to novel agents identified by the screening assays described above and their use for treatment as described herein.

Detection Assays Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in many ways as polynucleotide reagents. For example, without limitation, these sequences may be used to (i) identify an individual from a small amount of biological sample (tissue typing); and (ii) aid in forensic identification of biological samples. . Some of these applications are described in the sections below.

Tissue Typing The NOVX sequences of the present invention can be used to distinguish individuals from a small biological sample. In this technique, genomic DNA of an individual is digested with one or more restriction enzymes and probed on Southern blots to generate unique bands for identification. The sequences of the present invention are based on RFLP (US Pat. No. 5,272,0).
Useful as an additional DNA marker for the "restriction fragment length polymorphism" described in No. 57).

In addition, the sequences of the present invention may be used to provide an alternative technique for determining the DNA sequence for each actual base for selected portions of the individual's genome. Therefore, using the NOVX sequence described herein, two P's from the 5'and 3'ends of the sequence are used.
CR primers can be prepared. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

The panel of corresponding DNA sequences from individuals prepared in this way was
Having a unique set of such DNA sequences due to allelic differences,
It may provide unique identification. The sequences of the invention can be used to obtain such discriminating sequences from individuals and tissues. The NOVX sequences of the present invention uniquely represent portions of the human genome. Allelic variations occur to some extent in the coding regions of these sequences and to a greater extent in non-coding regions. It is estimated that allelic variation between individual humans occurs approximately once every 500 bases. The high degree of allelic variation is due to single nucleotide polymorphisms (SNPs), including restriction fragment length polymorphisms (RFLPs).

Each of the sequences described herein can, to some extent, be used as a standard against which DNA from an individual can be compared for purposes of discrimination. Not so many sequences are required to distinguish individuals, as more polymorphisms occur in non-coding regions. Non-coding sequences can comfortably provide positive individual identification, possibly with a panel of 10-1,000 primers. These primers each yield an amplified non-coding sequence of 100 bases. If a putative coding sequence is used (eg, the sequence in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21), a more suitable number of primers for positive individual identification is , 500 to 2,000.

Predictive Medicine The present invention also provides diagnostic assays, prognostic assays, drug genomes.
Genomics) and monitoring clinical trials relate to the field of predictive medicine, where prognostic treatment is used for prognostic (predictive) purposes, thereby treating individuals prophylactically. Accordingly, one aspect of the present invention is the expression of NOVX proteins and / or nucleic acids, and the activity of NOVX in a biological sample (eg, blood, serum, cells, tissues).
In relation to aberrant NOVX expression or activity,
Determine whether an individual suffers from a disease or disorder, or is at risk of developing a disorder. Disorders associated with abnormal NOVX expression activity include, for example:
Abnormal cell proliferation, cell differentiation, and cell migration (eg, cancer, angiogenesis and wound healing), neurological disorders (eg, paraneoplastic neurological disorders, accidental ataxia,
Characterized by autosomal dominant myocardial pulsatility, stroke, Parkinson's and Alzheimer's disease, enamel deficiency (eg, enamel hypoplasia) and inappropriate proteolysis (eg, atherosclerosis and abdominal aortic aneurysm) There are obstacles that can be attached.

The present invention also provides a prognostic (or predictive) assay for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in the NOVX gene can be assayed in biological samples. Such an assay may be used for prognostic or predictive purposes, thereby preventing an individual prior to the onset of a disorder characterized by or associated with the expression of NOVX protein, nucleic acid or biological activity. Treatment.

Another aspect of the invention provides a method for determining the expression or activity of NOVX protein, nucleic acid in an individual, whereby an appropriate therapeutic or prophylactic factor for that individual (herein disclosed). (Referred to as "pharmacogenomic genome"). A pharmacogenomic is based on an individual's genotype (eg, an individual's genotype tested to determine an individual's ability to respond to certain factors) for therapeutic or prophylactic treatment of the individual. Allows selection of factors (eg drugs).

Yet another aspect of the invention relates to monitoring the effects of factors (eg, drugs, compounds) on NOVX expression or activity in clinical trials.

[0280]   These and other factors are described in further detail in the sections below.

Diagnostic Assays An exemplary method for detecting the presence or absence of NOVX in a biological sample is the step of obtaining a biological sample from a test subject, and treating the biological sample with NOVX. Nucleic acid encoding a protein or NOVX protein (
Contacting with a compound or agent capable of detecting (eg, mRNA, genomic DNA) such that the presence of NOVX is detected in the biological sample,
Including steps. The agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA. This nucleic acid probe is, for example, a full-length NOVX nucleic acid (for example, SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 1).
9 or 21 or portions thereof nucleic acids (eg, at least 15, 30, 50
, 100, 250 or 500 nucleotides in length,
And a nucleic acid sufficient to specifically hybridize with NOVX mRNA or genomic DNA under stringent conditions)). Other suitable probes for use in the diagnostic assays of the invention are described herein.

One agent for detecting NOVX protein is an antibody capable of binding NOVX, preferably an antibody with a detectable label. Antibodies to the proteins of the invention may be used in methods known in the art relating to protein localization and / or quantification (eg for use in measuring the level of a protein in a suitable physiological sample). , For use in diagnostic methods, for use in protein imaging, etc.). In certain embodiments, antibodies to proteins or derivatives, fragments, analogs or homologs thereof that contain an antigen binding domain are used as the pharmacologically active composition.

Antibodies specific for the proteins of the invention can be used to isolate the protein by standard techniques, such as immunoaffinity chromatography or immunoprecipitation. Such antibodies may facilitate the purification of native protein antigens from cells and the recombinantly produced antigens expressed in host cells. further,
Such antibodies can be used to detect antigenic proteins (eg, in cell lysates or cell supernatants) in order to assess the amount and pattern of expression of the antigenic protein. Antibodies to this protein can be used, for example, to diagnostically monitor protein levels in tissues as part of a clinical trial procedure to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (ie, physically linking) the antibody to a detectable substance.
Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances,
Bioluminescent and radioactive substances are mentioned. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of such fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine and dichlorotriazinylamine (dichlor).
fluorescein, dansyl chloride or phycoerythrin; examples of luminescent substances include luminol; examples of bioluminescent substances include luciferase, luciferin and aequorin, and examples of suitable radioactive substances. ^{Include 125} I, ¹³¹ I, ³⁵ S or ³ H.

The antibody may be polyclonal or, more preferably, a monoclonal antibody. Intact antibodies or fragments thereof (eg Fab or F (ab ′) ₂ ) can be used. The term "labeled" with respect to a probe or antibody refers to direct labeling of the probe or antibody by coupling (ie, physically linking) a detectable substance to the probe or antibody. , As well as indirectly labeling the probe or antibody with its reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of the primary antibody with a fluorescently labeled secondary antibody, and end labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject as well as tissues, cells and fluids present in the subject. That is, the detection method of the present invention can be used to detect NOVX mRNA, protein or genomic DNA in a biological sample in vitro and in vivo. For example, NOV
In vitro techniques for detection of X mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for the detection of NOVX proteins include enzyme-linked immunosorbent assay (
ELISA), Western blot, immunoprecipitation and immunofluorescence. N
In vitro techniques for detecting OVX genomic DNA include Southern hybridizations. In addition, in vivo techniques for detection of NOVX proteins include introducing a labeled anti-NOVX antibody into the subject. For example, the antibody can be labeled with a radioactive marker. The presence and location of radioactive markers in this subject can be detected by standard imaging techniques.

[0285] In one embodiment, the biological sample comprises protein molecules from the test subject. Alternatively, the biological sample may include mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.

In one embodiment, the method of the present invention further comprises obtaining a control biological sample from a control subject, the control sample being a NOV.
In contact with a compound or agent capable of detecting X protein, mRNA or genomic DNA such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and in a control sample thereof. Presence of NOVX protein, mRNA or genomic DNA and NOVX protein, mRNA or genomic D in the test sample
Comparing with the presence of NA.

The present invention also includes a kit for detecting the presence of NOVX in a biological sample. For example, the kit may include: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; a means for determining the amount of NOVX in the sample; and in the sample A means for comparing the amount of NOVX with a standard. The compound or agent can be packaged in a suitable container. This kit is
Instructions for using the kit to detect OVX protein or nucleic acid may be provided.

Prognosis Assays The diagnostic methods described herein may be further utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant expression or activity of NOVX. Can be identified. For example, an assay described herein (eg, the diagnostic assay described above or the assay described below) can be utilized to have or be at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. A subject having can be identified. Such disorders include, for example, abnormal cell proliferation, cell differentiation, and cell migration (eg, cancer, angiogenesis and wound healing), neurological disorders (eg, paraneoplastic neurological disorders, contingencies). Ataxia, autosomal dominant myocarditis, seizures, Parkinson's disease and Alzheimer's disease), enamel deficiency (
For example, enamel hypoplasia) and disorders characterized by inappropriate proteolysis (eg, atherosclerosis and abdominal aortic aneurysm).

Alternatively, this prognostic assay may be utilized to identify subjects who have or are at risk of developing a disease or disorder. Accordingly, the invention provides methods for identifying diseases or disorders associated with aberrant expression or activity of NOVX. Here, a test sample is obtained from the subject and NOVX protein or nucleic acid (eg, mRNA, genomic DNA) is detected, wherein NOVX is
The presence of the X protein or nucleic acid is a diagnostic indicator for a subject having or at risk of developing a disease or disorder associated with aberrant expression or activity of NOVX. As used herein, "test sample" refers to a biological sample obtained from a subject of interest. For example, the test sample can be a biological fluid (eg, serum), cell sample, or tissue.

In addition, the prognosis assay described herein can be used to provide a subject with an agent (eg, agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate). Whether one can administer to treat a disease or disorder associated with aberrant expression or activity of NOVX can be determined. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder. Accordingly, the invention provides a method for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant expression or activity of NOVX. Here, a test sample is obtained and NOVX protein or nucleic acid is detected (eg, where the presence of NOVX protein or nucleic acid is associated with aberrant expression or activity of NOVX upon administration of the agent). Is a diagnostic indicator for subjects in which the disorder may be treated).

The methods of the invention also detect genetic damage in the NOVX gene, whereby a subject carrying the damage gene is at risk for a disorder characterized by aberrant cell proliferation and / or differentiation. It can also be used to determine whether or not. In various embodiments, the method provides for the presence or absence of a genetic lesion in a sample of cells from the subject characterized by at least one alteration that affects the integrity of the gene encoding the NOVX protein, Alternatively, the step of detecting misexpression of the NOVX gene is included. For example, such genetic damage can be detected by confirming the presence of at least one of: (i) a deletion of one or more nucleotides from the NOVX gene; (ii).
(Iii) addition of one or more nucleotides to the NOVX gene; (iii) substitution of one or more nucleotides of the NOVX gene, (iv) chromosomal rearrangement of the NOVX gene;
v) alterations in the level of messenger RNA transcripts of the NOVX gene, (v
i) abnormal alteration of NOVX gene (for example, abnormal alteration of methylation pattern of genomic DNA), (vii) presence of non-wild type splicing pattern of messenger RNA transcript of NOVX gene, (viii) non-wild type level of NOVX protein , (Ix) allelic deletion of the NOVX gene, and (x) inappropriate post-translational modification of the NOVX protein. As described herein, there are a number of known assay techniques in the art that can be used to detect damage in the NOVX gene. A preferred biological sample is a peripheral blood leukocyte sample isolated from a subject by conventional means. However, any biological sample containing nucleated cells can be used, including, for example, buccal mucosal cells.

In certain embodiments, the detection of damage is performed by polymerase chain reaction (PCR) (
See, eg, US Pat. Nos. 4,683,195 and 4,683,202) (eg, anchor PCR or RACE PCR), or ligation chain reaction (LCR) (eg, Landegran et al. (1988). ) Scien
ce 241: 1077-1080; and Nakazawa et al. (1994) P.
roc. Natl. Acad. Sic. USA 91: 360-364)). The latter may be particularly useful for detecting point mutations in the NOVX gene) (see Abravaya et al., 1995 Nucl Acids Res 23: 675-682). The method comprises collecting a sample of cells from a patient, isolating a nucleic acid (eg, genome, mRNA or both) from the cells of the sample, NO
Contacting one or more primers that specifically hybridize to the VX gene with its nucleic acid sample under conditions such that hybridization and amplification of the NOVX gene (if present) occurs, and the presence of an amplification product Alternatively, it may include detecting its absence, or detecting the size of its amplification product and comparing its length with a control sample. It is envisioned that PCR and / or LCR may be desirably used as a preliminary amplification step with any of the techniques used to detect the mutations described herein.

Alternative amplification methods include: self-sustaining sequence replication (Guat).
elli et al., 1990, Proc. Natl. Acad. Sci. USA 87
1874-1878), a transcription amplification system (Kwoh, et al., 1989,
Proc. Natl. Acad. Sci. USA 86: 1173-1177), Qβ replicase (Lizardi et al., 1988, BioTec.
hnology 6: 1197), or any other nucleic acid amplification method, followed by detection of the amplified molecule using techniques well known to those of skill in the art. These detection schemes are particularly useful for the detection of nucleic acid molecules when such molecules are present in very low numbers.

In an alternative embodiment, mutations in the NOVX gene from sample cells can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (if necessary), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. To be done. The difference in size of fragment length between sample DNA and control DNA is
The mutation in A is shown. In addition, the use of sequence-specific ribozymes (see, eg, US Pat. No. 5,493,531) can be used to score for the presence of specific mutations by the occurrence or loss of ribozyme cleavage sites. .

In another embodiment, a gene mutation in NOVX is the hybridization of a sample nucleic acid and a control nucleic acid (eg, DNA or RNA) to a high density array containing hundreds or thousands of oligonucleotide probes. Can be identified by (eg Cronin et al., 1996, Human Mut.
ation 7: 244-255; Kozal et al., 1996, Nat. Med.
2: 753-759). For example, the gene mutation in NOVX is
It can be identified in a two-dimensional array containing photogenerated DNA probes as described in Cronin et al., Supra. Briefly, the first hybridization array of probes was used to identify base changes between sequences by scanning over a long stretch of DNA in the sample and control to create a linear array of probes with overlapping sequences. obtain. This step allows the identification of point mutations. This step involves the formation of a second hybridization array, which allows the characterization of specific mutations with a smaller specialized probe array complementary to all variants or variants detected. Continue. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, the NOVX gene can be sequenced directly using any of a variety of sequencing reactions known in the art, and the sample NOVX sequence and its corresponding wild type (control). 3.) Mutations can be detected by comparison with the sequence. Examples of sequencing reactions are Maxim and Gilbert (
1977) Proc. Natl. Acad. Sci. USA 74: 560 or Sanger (1977) Proc. Natl. Acad. Sci. USA
74: 5463, which is based on the technology developed by It is also contemplated that any of a variety of automated sequencing procedures may be utilized when performing diagnostic assays (eg, Naeve et al., (1995) Biotechnique).
s 19: 448). These include sequencing by mass spectrometry (
For example, PCT International Publication Number WO 94/16101; Cohen et al. (1996).
) Adv. Chromatography 36: 127-162; and Gr.
ifin et al. (1993) Appl. Biochem. Biotechnol.
38: 147-159).

[0297] Other methods for detecting mutations in the NOVX gene include using protection from cleavage agents to detect mismatched bases in RNA / RNA or RNA / DNA heteroduplexes. . For example, Myers et al. (19
85) Science 230: 1242. In general, the art of "mismatch cleavage" uses RNA that comprises (labeled) wild-type NOVX sequences.
Alternatively, it begins with the step of providing a heteroduplex formed by hybridizing DNA with a potential mutant RNA or mutant DNA obtained from a tissue sample. Treating this double-stranded duplex with an agent that cleaves the single-stranded region of the duplex (eg, which is present due to a base pair mismatch between its control and sample strands) To do. For example, RNA / DNA duplex is
and the DNA / DNA hybrid can be treated with S ₁ nuclease to enzymatically digest its mismatched regions. In other embodiments, either DNA / DNA or RNA / DNA duplexes may be treated with hydroxylamine or osmium tetroxide and with piperidine to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then size-separated on a denaturing polyacrylamide gel to determine the site of mutation. For example, Cotton et al. (1988) Proc. N
atl. Acad. Sci. USA 85: 4397; Saleeba et al. (19).
92) Methods Enzymol 217: 286-295. In one embodiment, the control DNA or RNA can be labeled for detection.

In yet another embodiment, the mismatch cleavage reaction involves NOVX obtained from a sample of cells with one or more proteins that recognize mismatched base pairs in double-stranded DNA (so-called “DNA mismatch repair” enzymes). Used in defined systems to detect and map point mutations in cDNA. For example,
E. E. coli mutY enzyme cleaves A at a G / A mismatch and HeL
Thymidine DNA glycosidase from a cell cleaves T at a G / T mismatch. For example, Hsu et al. (1994) Carcinogenesis 15:16.
57-1662. According to an exemplary embodiment, probes based on NOVX sequences (eg, wild type NOVX sequences) are hybridized to cDNA or other DNA products from test cells. The duplex is treated with a DNA mismatch repair enzyme and its cleavage product (if any) can be detected, such as by electrophoresis protocols. See, eg, US Pat. No. 5,459,039.

[0299] In another embodiment, alterations in electrophoretic mobility are used to identify mutations in the NOVX gene. For example, single-stranded conformational polymorphisms (SSCPs) can be used to detect differences in electrophoretic mobilities between mutant and wild-type nucleic acids (eg, Orita et al. (1989) Proc. N
atl. Acad. Sci. USA: 86: 2766; Cotton (1993).
) Mutat. Res. 285: 125-144; Hayashi (1992).
Genet. Anal. Tech. Appl. 9: 73-79).
Single-stranded DNA fragments of sample and control NOVX nucleic acids are denatured and reconstituted. The secondary structure of single-stranded nucleic acids varies according to sequence, and the resulting changes in electrophoretic mobility even allow the detection of single base changes. D
The NA fragment may be labeled or detected with a labeled probe. The sensitivity of the assay can be enhanced by using RNA (rather than DNA) whose secondary structure is more sensitive to changes in sequence. 1
In one embodiment, the methods of the invention utilize heteroduplex analysis to separate double-stranded heteroduplex molecules based on changes in electrophoretic mobility. For example, Keen et al. (1991) Trends Genet. See 7: 5.

In yet another embodiment, migration of mutant or wild type fragments in a polyacrylamide gel containing a constant gradient of denaturing agent is assayed using denaturing gradient gel electrophoresis (DGGE). For example, Myers et al. (1985) N
See ature 313: 495. When DGGE is used as the method of analysis, the DNA can be, for example, by PCR a high melting point GC-rich DN of about 40 bp.
It is modified by adding a GC clamp of A to ensure that it is not completely denatured. In a further embodiment, temperature gradients are used instead of denaturant gradients to identify differences in the mobility of control and sample DNA. For example, Rosenbaum and Reissner (198
7) Biophys. Chem. 265: 12753.

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers are
Known mutations can be prepared to be centrally located and then hybridized to the target DNA under conditions that allow hybridization only if a perfect match is found. For example, Saiki et al. (1986) Nature 32.
4: 163); Saiki et al. (1989) Proc. Natl. Acad. Sc
i. USA 86: 6230. Such allele-specific oligonucleotides hybridize to PCR amplified target DNA or many different mutations when the oligonucleotide is attached to a hybridizing membrane and hybridized to labeled target DNA. To be done.

Alternatively, allele-specific amplification techniques that rely on selective PCR amplification can be used in conjunction with the present invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification is
Depends on differential hybridization; see, for example, Gibbs et al. (1989).
Nucl. Acids Res. 17: 2437-2448), or
Alternatively, it may carry the mutation of interest at the 3'-most end of one primer, which may inhibit mismatches or reduce polymerase extension under appropriate conditions (eg, Prosner (1993) Tibtech. 11: 238). checking). Furthermore, introducing new restriction sites in the region of mutations may be desirable for developing cleavage-based detection. For example, Gasparini et al.
1992) Mol. See Cell Probes 6: 1. It is anticipated that in certain embodiments, amplification may also be performed using Taq ligase for amplification. For example, Barany (1991) Proc. Natl.
Acad. Sci. See USA 88: 189. In such cases, ligation will only occur if there is an exact match at the 3'end of the 5'sequence and by searching for the presence or absence of amplification the presence of a known mutation at a particular site. Allows to detect.

The methods described herein can be performed, for example, by utilizing a pre-packaged diagnostic kit that includes at least one probe nucleic acid or antibody reagent described herein. , Which can conveniently be used, for example, in a clinical setting for diagnosing patients with symptoms or familial history of a disease or disorder involving the NOVX gene.

In addition, any cell type or tissue in which NOVX is expressed, preferably peripheral blood leukocytes, can be utilized in the prognosis assay described herein. However, any biological sample containing nucleated cells (eg, including buccal mucosal cells)
Can be used.

Pharmacogenomics Factors, or modulators, that have a stimulatory or inhibitory effect on NOVX activity (eg, NOVX gene expression) are identified by the screening assays described herein. As such, disorders characterized by abnormal cell proliferation, cell differentiation and cell migration (eg, cancer, angiogenesis and wound healing), neurological disorders (eg, paraneoplastic neurological disorders, episodic). Ataxia (ep
isoditic ataxia), autosomal dominant myokimia, seizures, Parkinson's disease and Alzheimer's disease), enamel defect
(Eg enamel hypoplasia) as well as inappropriate proteolysis (eg,
Atherosclerosis and abdominal aortic aneurysm
aneurisms)) to treat (prophylactically or therapeutically) an individual can be administered to an individual. In conjunction with such treatment, an individual's pharmacogenomics, ie, a study of the relationship between an individual's genotype and that individual's response to foreign compounds or drugs, may be considered. Differences in metabolism of therapeutics can lead to severe toxicity and therapeutic failure by altering the relationship between dose and blood concentration of the pharmacologically active drug. Therefore, the pharmacogenomics of an individual allows for the selection of effective agents (eg, drugs) for prophylactic or therapeutic treatment based on consideration of the individual's genotype. Such pharmacogenomics can further be used to determine the appropriate dosage and therapeutic agent regimen. Therefore, NOV
The activity of the X protein, the expression of NOVX nucleic acid, or the mutation content of the NOVX gene in the individual can be determined, thereby selecting the appropriate agent for therapeutic or prophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug properties and aberrant effects in affected individuals. For example, Eichelbaum, 1996, Clin. Exp. Phar
macol. Physiol. , 23: 983-985; Linder, 199.
7, Clin. Chem. , 43: 254-266. In general, two types of pharmacogenomic states can be distinguished. Genetic status is transmitted as one factor that modifies the way a drug acts on the body (altered drug action), or genetic status is one factor that modifies the way the body acts on a drug. Is transmitted as (altered drug metabolism). These pharmacogenomic states can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common hereditary enzymatic disease, the main clinical complications of which are oxidative drugs (antimalarials, sulfonamides, analgesics, nitrofuran). ) Ingestion and consumption of broad beans.

In an exemplary embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms in drug-metabolizing enzymes, such as N-acetyltransferase 2 (NAT2) and cytochrome P450 enzymes CYP2D6 and CYP2C19, may indicate that some patients do not have the expected drug effect, or standard And provided explanations regarding excessive drug response and severe toxicity after ingestion of safe doses of the drug. These polymorphisms are associated with two phenotypes in the population, those with high metabolic capacity.
(EM) and poor metabolizing capacity (poor metabol)
It is expressed by an (izer) (PM)). The prevalence of PM varies between different populations. For example, the gene encoding CYP2D6 is highly polymorphic, and some mutations have been identified in PM, all leading to the absence of functional CYP2D6. Those with low metabolic capacity for CYP2D6 and CYP2C19 quite often experience exaggerated drug responses and side effects when they receive standard doses. When the metabolite is the active therapeutic moiety, PM does not show a therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-forming metabolite, morphine. The other extreme are the so-called ultra-rapid metabolizers who do not respond to standard doses. Molecules that are the basis for ultrarapid metabolism have recently been identified to be due to CYP2D6 gene amplification.

Accordingly, the activity of the NOVX protein, the expression of the NOVX nucleic acid, or the mutational content of the NOVX gene in an individual may be determined, thereby selecting the appropriate agent for therapeutic or prophylactic treatment of that individual. . In addition, pharmacogenomic studies can be used to apply the genotypes of polymorphic alleles encoding drug-metabolizing enzymes for identification of an individual's drug-responsive phenotype. This finding, when applied to dose or drug selection, may avoid adverse reactions or treatment failures, thus reducing NO in subjects.
Therapeutic or prophylactic efficacy may be enhanced when treated with a modulator of VX (eg, a modulator identified by one of the exemplary screening assays described herein).

Monitoring Effects During Clinical Trials Monitoring the effect of a drug (eg, drug, compound) on NOVX expression or activity (eg, ability to regulate aberrant cell growth) is a basic drug It can be applied not only to screening but also to clinical trials. For example, the NOVX of a drug determined by a screening assay as described herein.
Gene expression, potency to increase protein levels, or potency to upregulate NOVX activity can be monitored in clinical trials in subjects exhibiting reduced NOVX gene expression, protein levels, or downregulated NOVX activity. . Alternatively, the efficacy of an agent as determined by a screening assay in reducing NOVX gene expression, protein levels, or down-regulating NOVX activity may result in increased NOVX gene expression, protein levels, or up-regulated NOVX activity. It may be monitored in clinical trials of the subject indicated. In such clinical trials, the expression or activity of NOVX, and preferably other genes involved in, for example, cell proliferation or immune disorders, is "read out," that is to say in particular cells. It can be used as a marker of immune responsiveness.

By way of example, but not of limitation, the gene containing NOVX (which is N
That OVX activity (eg, identified in a screening assay as described herein) is modulated intracellularly by treatment with an agent (eg, compound, drug or small molecule), Can be identified. Thus, to study the effects of agents on cellular proliferative disorders, cells can be isolated and RNA can be prepared and expression of NOVX and other genes involved in this disorder, eg, in clinical trials. It can be analyzed for levels. Level of gene expression (
That is, the gene expression pattern) is described herein by Northern blot analysis or RT-PCR, as described herein, or by measuring the amount of protein produced. Or by measuring the level of activity of NOVX or other genes.
Can be quantified. In this manner, the gene expression pattern can act as a marker that is indicative of the physiological response of cells to the drug. Thus, the response state can be determined prior to treatment of the individual with the agent, and at various times during the treatment.

In one embodiment, the invention is a drug (eg, agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other identified by a screening assay described herein). Of drug candidates) for monitoring the efficacy of treatment in a subject, which comprises:
Comprising the steps of: (i) obtaining a pre-dose sample from the subject prior to administration of the drug; (ii) in the pre-dose sample, NOVX protein, mR
Detecting the level of expression of NA, or genomic DNA; (iii) obtaining one or more post-dose samples from this subject; (iv) NOVX protein, mRNA, or genomic DNA in the post-dose sample. (V) the level of expression or activity of NOVX protein, mRNA, or genomic DNA in this pre-administration sample is compared with the level of expression or activity of NOVX protein, mRNA, or genomic DNA in this post-administration sample. Comparing the level of expression or activity of the agent; and (vi) altering the administration of the agent to this subject accordingly. For example, increased administration of the agent may be desirable to increase NOVX expression or activity (ie, increase the efficacy of the agent) to a level that is higher than detected. Alternatively, reduced administration of the agent may be desirable to reduce NOVX expression or activity to a level below that detected (ie, reduce efficacy of the agent).

Methods of Treatment The present invention is at risk for disorders associated with abnormal NOVX expression or activity (
Or susceptibility), or both prophylactic and therapeutic methods of treating a subject having this disorder. Disorders associated with aberrant NOVX expression include, for example, disorders characterized by aberrant cell proliferation, cell differentiation and cell migration (eg, cancer, angiogenesis and wound healing), neurological disorders (eg, tumors). Concomitant neurological disorders, episodic ataxia, autosomal dominant myokimia, seizures, Parkinson's disease and Alzheimer's disease, enamel deficiency (eg, enamel hypoplasia) and inappropriate proteolysis (eg, atherosclerosis) Disease and abdominal aortic aneurysm). These treatment methods are discussed more fully below.

Diseases and Disorders Diseases and disorders characterized by increased levels or biological activity (compared to a subject not afflicted with the disease or disorder) antagonize activity (
That is, it can be treated with a therapeutic agent (which reduces or inhibits). A therapeutic agent that antagonizes activity can be administered in a therapeutic or prophylactic manner. Therapeutic agents that may be utilized include, but are not limited to, (i) the peptides described above,
Or an analog, derivative, fragment or homolog thereof; (ii) an antibody against the above peptide; (iii) a nucleic acid encoding the above peptide; (iv)
Antisense nucleic acids and "dysfunctional" that are utilized to "knock out" the endogenous function of the peptide by homologous recombination (ie, for heterologous insertions within the coding sequence of the coding sequence for the peptide. Administration of nucleic acids (due to), eg, Capecchi, 1989, Science 244: 1288.
~ 1292); or (v) modulators (ie, inhibitors, agonists and antagonists (additional peptidomimetics of the invention or the invention) that alter the interaction between the peptide described above and its binding partner. Antibody specific for the peptide)).

Diseases and disorders characterized by decreased levels or biological activity (compared to a subject not afflicted with the disease or disorder) have increased activity (ie, are agonists of the activity). It can be treated with a therapeutic agent. A therapeutic that upregulates activity can be administered in a therapeutic or prophylactic manner. Therapeutic agents that may be utilized include, but are not limited to, the peptides described above, or analogs, derivatives, fragments or homologs thereof; or agonists that increase bioavailability.

Increased or decreased levels can be readily detected by quantifying peptide and / or RNA. This quantification involves obtaining a tissue sample of a patient (eg, from a biopsy tissue) and obtaining the sample at the RNA or peptide level of the expressed peptide (or mRNA of the above peptide),
By assaying structure and / or activity in vitro. Methods well known in the art include, but are not limited to, immunoassays (eg, by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.).
And / or hybridization assays to detect mRNA expression (eg, Northern assays, dot blots, in situ hybridization, etc.).

Prophylactic Method In one aspect, the invention provides for a disease or condition associated with aberrant NOVX expression or activity in a subject, including expression of NOVX or at least one NO.
Methods for preventing by administering to the subject an agent that modulates VX activity are provided. A subject at risk of developing a disease caused by or caused by aberrant NOVX expression or activity is, for example, by any of the diagnostic or prognostic assays described herein, or a combination thereof. , Can be identified. Administration of a prophylactic drug will prevent the disease or disorder from
Alternatively, it may be delayed before its appearance before the symptoms that are characteristic of this NOVX abnormality. Depending on the type of NOVX abnormality, for example, a NOVX agonist drug or NOVX antagonist drug can be used to treat the subject. The appropriate agent can be determined based on the screening assays described herein. The prevention method of the present invention is further discussed in the following subsections.

Method of Treatment Another aspect of the invention relates to a method of modulating NOVX expression or activity for therapeutic purposes. The modulating methods of the invention include contacting a cell with an agent that modulates one or more of the activities of NOVX protein activity for that cell.
Agents that modulate NOVX protein activity include nucleic acids or proteins, naturally occurring cognate ligands of NOVX proteins, peptides, NOVX peptidomimetics,
Or it may be an agent as described herein, such as other small molecules. 1
In one embodiment, the agent stimulates one or more of NOVX protein activities. Examples of such stimulatory agents include active NOVX proteins and NOVX-encoding nucleic acid molecules introduced into the cell. In another embodiment, the agent inhibits one or more of NOVX protein activities. Examples of such inhibitory agents include antisense NOVX nucleic acid molecules, and anti-NOVX antibodies. These methods of regulation are in vitro (eg,
It can be performed by culturing the cells with the agent) or in vivo (eg, by administering the agent to a subject). in this way,
The invention provides methods of treating an individual suffering from a disease or disorder characterized by aberrant expression or activity of a NOVX protein or NOVX nucleic acid molecule. In one embodiment, the method modulates (eg, upregulates or downregulates) NOVX expression or activity.
Administering an agent (eg, an agent identified by the screening assays described herein) or a combination of such agents. In another embodiment, the method comprises administering a NOVX protein or nucleic acid molecule as a treatment to compensate for reduced or aberrant NOVX expression or activity.

Stimulation of NOVX activity is desirable in situations where NOVX is abnormally downregulated, and / or where increased NOVX activity appears to have a beneficial effect. One example of such a situation is where the subject is a disorder characterized by abnormal cell proliferation and / or cell differentiation (eg, cancer or immune related).
Is the case. Another example of such a situation is when a subject has an immunodeficiency disease (eg, AIDS).

The antibodies of the present invention, including polyclonal antibodies, monoclonal antibodies, humanized antibodies, and fully human antibodies can be used as therapeutic agents. Such drugs
Generally used to treat or prevent diseases and pathologies in a subject. An antibody preparation, preferably one with high specificity and high affinity for its target antigen, is administered to a subject and generally has an effect due to its binding to its target. Such effects can be of one of two types, depending on the specific nature of the interaction between a given antibody molecule and the target antigen of interest. In the first example, administration of the antibody may prevent or inhibit binding of the target to the endogenous ligand to which it naturally binds. In this case, the antibody binds to the target and masks the binding site of the naturally occurring ligand, where that ligand acts as an effector molecule. Thus, receptors mediate the signaling pathways whose ligands are responsible for signaling.

Alternatively, the effect may be that the antibody elicits a physiological result by binding to an effector binding site on the target molecule. In this case, the target (receptor with an endogenous ligand that may be absent or defective in disease or pathology)
Binds the antibody as a surrogate effector ligand to initiate a receptor-based signaling event by the receptor.

A therapeutically effective amount of an antibody of the invention generally refers to the amount required to achieve a therapeutic purpose. As mentioned above, this can be a binding interaction between the antibody and its target antigen that in some cases interferes with the function of the target and in others promotes a physiological response. The amount required for administration will further depend on the binding affinity of the antibody for its specific antigen, and also, the amount will be such that the antibody administered is from the free volume of the subject to which the antibody is administered. Depends on the rate of depletion. A general range for a therapeutically effective dosage of an antibody or antibody fragment of the invention is, as a non-limiting example, from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Common dosing frequencies can range, for example, from twice daily to once a week.

Determining Biological Effects of Therapeutic Agents In various embodiments of the invention, suitable in vitro or in vivo assays are performed to determine the effect of a particular therapeutic agent and its administration is indicative of treatment of diseased tissue. Decide whether or not.

In various specific embodiments, in vitro assays are performed with a representative cell type involved in the disorder of the patient and whether a given therapeutic agent exerts the desired effect on this cell type. Can decide. The compounds used in therapy may be tested in suitable animal model systems before testing in human subjects. These animal model systems include, but are not limited to: rat, mouse, chicken, cow, monkey, rabbit and the like. Similarly, for in vivo testing, any animal model system known in the art can be used prior to administration to human subjects.

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Examples Example 1: Methods of Identifying Nucleic Acids of the Invention Novel nucleic acid sequences have been made available by GenBank to Genomic.
CuraGen Corporate run against Dairy Files
identified by TblastN using the Ion sequence file. Nucleic acids were further predicted by the program GenScan ^™ , which included exon selection. These were further modified by similarity using the BLAST search. The sequences were then manually corrected for apparent discrepancies, resulting in sequences encoding the full-length protein.

Example 2: Identification of NOV3 (CG51785-06) The sequence of accession number CG51785-06 was obtained by laboratory cloning of a cDNA fragment by predicting the sequence in silico. A cDNA fragment covering either the full-length DNA sequence, or part of that sequence, or both was cloned. The in silico predictions are based on sequences available to Curagen in its own database of sequences or databases of public human sequences, and either full-length DNA sequences or some portion thereof are provided.

Laboratory cloning was performed using one or more of the methods summarized below: SeqCalling ^™ Technology: cDNAs for multiple tissue types, normal and disease states, physiological states, and different donors. Obtained from various human samples, which represent the embryological state of origin. Samples were obtained as whole tissue, primary cells or tissue culture primary cells or cell lines. Cells and cell lines can be treated with biological or chemical agents that regulate gene expression, such as growth factors, chemokines, or steroids. Next, the obtained cDNA was processed by CuraGen's original SegCal.
Sequencing was performed using the Ling technique. Sequence traces were manually evaluated and, where appropriate, edited for correction. Cd from all samples
The NA sequences are assembled together (sometimes including the official human sequences) to generate a consensus sequence for each assembly using a bioinformatics program. Include each assembly in the CuraGen Corporation database. The sequence has a degree of identity with another component of at least 9 over 50 bp.
It was included as an ingredient for the assembly when it was 5%. Each assembly represents a gene or portion thereof, and variants (eg, single nucleotide polymorphisms (
SNP), insertions, deletions, and splicing to form other sequence variants).

Exon ligation: The cDNA encoding the CG51785-06 sequence was cloned by polymerase chain reaction (PCR) using the following primers: 5'-TCTCCCCAGAGGCCAGGAC-3 '(SEQ ID NO: 59) and 5'-CGCATGGTTTTGGGGATTTG. -3 '(SEQ ID NO: 60).

Primers were designed based on the in silico predictions of full length or some portion (one or more exons) of the cDNA / protein sequences of the invention. Using these primers, cDNA from a pool containing expressed human sequences derived from the following tissues
Amplified NA: adrenal gland, bone marrow, brain-tonsils, brain-cerebellar, brain-hippocampus, brain-substantia nigra, brain-
Thalamus, brain-whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma-large, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord,
Spleen, stomach, testis, thyroid, trachea, and uterus.

Multiple clones were sequenced and these fragments were pooled together and (
Bioinformatics programs are used to generate consensus sequences for each assembly (sometimes including public human sequences). Cu each assembly
Included in raGen Corporation database. A sequence is given a degree of identity with another component of at least 95% over 50 bp,
It was included as a component for the assembly. Each assembly represents a gene or portion thereof and contains information for variants, such as splicing to form single nucleotide polymorphisms (SNPs), insertions, deletions, and other sequence variants.

Physical clone: The PCR product derived by exon ligation covering the entire open reading frame was cloned into the pCR2.1 vector from Invitrogen to produce clone 27824582_0_105.69849.
6. F5 provided.

Variant sequences are also included in this application. The variant sequence is a single nucleotide polymorphism (
SNP). In some examples, a SNP may be referred to as a "cSNP" to indicate that the nucleotide sequence containing the SNP is of cDNA origin. SNPs can occur in several ways. For example, a SNP can result from the substitution of one nucleotide for another at a polymorphic site. Such substitutions can be either rearrangements or transversions. SNPs can also result from nucleotide deletions or nucleotide insertions with respect to the reference allele. In this case, polymorphic sites are sites where one allele creates a gap for a particular nucleotide in another allele. SN existing in the gene
P may result in a modification of the amino acid encoded by the gene at the SNP position. If the codon containing the SNP encodes the same amino acid as a result of the degeneracy of the genetic code, then the SNP within the gene may also be silent. SNPs that are outside the region of the gene or within the intron of the gene do not result in alterations in any amino acid sequence of the protein, but may result in altered control of the expression pattern. Examples include transient expression, regulation of physiological response, control of cell type expression, intensity of expression and alteration in transcriptional message stability.

The DNA and protein sequences for the novel collagen-like gene were obtained by exon ligation and are reported herein as NOV3 (CG51785-06).

Example 3. Molecular Cloning of NOV11 (AL096677_A) The putative mature form of the OV11 protein for clone AL096677_A is (SEQ ID NO: 22). That is, amino acid residue 20 to amino acid residue 14
The 8 regions were targeted for cloning. The PCR primers shown below were prepared.

[0335]

[Chemical 1] The PCR reaction was started using 5 ng of human testis cDNA template.
The reaction mixture contained 1 μM each of AL_096677_A in 50 μl reaction volume.
Mat-F and AL096677_A REV primer, 5 μmol d
NTP (Clontech Laboratories, Palo Alto
CA) and 1 μl of 50 × Advantage-HF 2 polymerase (
Clontech Laboratories, Palo Alto CA). The following reaction conditions were used: a) 96 ° C. for 3 minutes b) 96 ° C. for 30 seconds Denaturation c) 70 ° C. for 30 seconds Primer annealing: this temperature was gradually reduced at 1 ° C./cycle d) 72 ° C. for 3 minutes Repeat extension steps b to d 10 times e) 96 ° C. for 30 seconds Denaturation f) 60 ° C. for 30 seconds Annealing g) 72 ° C. for 3 minutes Repeat extension steps e to g 35 times h) 72 ° C. for 5 minutes Final extension.

Amplification products with the expected size of approximately 450 bp were detected by agarose gel electrophoresis. This fragment was purified from an agarose gel and the pCR2.1 vector (Invitrogen Carlsba was recommended according to the manufacturer's recommendations.
d, CA). This clone was cloned into pC2.1-AL096655_A-
It is referred to as S602-9B. The cloned insert was sequenced using the following sequence-specific primers.

[0337]

[Chemical 2] The nucleotide sequence obtained for clone pCR2.1_AL096655_A-S602-9B is shown in Table 34.

[0338]

[Table 35] The deduced corresponding amino acid sequence for clone pCR2.1_AL096655_A-S602-9B is shown in Table 35.

[0339]

[Table 36] There are 5 nucleotide changes (bold and underlined) that introduce 4 amino acid changes (bold and underlined) in the clone as compared to the sequence of the corresponding portion of clone AL096655_A. These are characterized in Tables 36 and 37. (Table 36. Comparison of the nucleotide sequence of clone AL09677_A (top row) with the nucleotide sequence of clone AL09677_A-S602-9B (bottom row))

[0340]

[Table 37] (Table 37. Amino acid sequence of clone AL09677_A (top row) and clone AL
(Comparison with the amino acid sequence of 09677_A-S602-9B (lower row))

[0341]

[Table 38] (Other Embodiments) Although the present invention has been described along with its detailed description, the above description is merely an example, and is not intended to limit the scope of the present invention. The scope of the invention is defined by the appended claims. Other aspects, advantages and modifications are within the scope of the above claims.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 9/00 A61P 17/02 4C085 9/10 101 25/00 4H045 17/02 25/16 25/00 25 / 28 25/16 35/00 25/28 43/00 111 35/00 C07K 14/47 43/00 111 16/18 C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12Q 1/02 1/21 1/68 A 5/10 C12P 21/08 C12Q 1/02 C12N 15/00 ZNAA 1/68 5/00 A // C12P 21/08 A61K 37/02 (31) Priority claim number 60/189, 139 (32) Priority date March 14, 2000 (March 14, 2000) (33) Country of priority claim United States (US) (31) Priority claim number 60/189, 140 (32) Priority date March 14, 2000 (March 14, 2000) (33) Country of priority claim United States (US) (31) Priority Right claim number 60 / 190,401 (32) Priority date March 17, 2000 (March 17, 2000) (33) Country of priority claim United States (US) (31) Priority claim number 60 / 190,231 (32) Priority date March 17, 2000 (March 17, 2000) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES , FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR , NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, C , CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE , SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Barnet, Coryne A .. M. United States Florida 32060, Gainesville, N. W. 43 Ardi Street 4830 Peenan Bar 253 (72) Inventor Fernandez, Elma United States Connecticut 06405, Branford, Florence Road 77 Number 2 Bee (72) Inventor Simquets, Richard A .. United States Connecticut 06516, West Haven, Riet Street 191 (72) Inventor Spaderna, Stephen Kay. United States Connecticut 06037, Berlin, Deerfield Drive 261 (72) Inventor Majunda, Kumu United States Connecticut 06905, Stanford, Silver Hill Lane 140 (72) Inventor Li, Li United States Connecticut 06410, Cheshire, Oak Street 478 F-Term (Reference) 4B024 AA01 AA11 BA53 BA61 BA80 CA04 CA07 CA09 CA12 CA20 DA01 DA02 DA03 DA05 DA11 DA12 FA02 GA11 HA11 HA13 HA14 HA15 HA17 4B063 QA01 QA05 QA19 QQ02 QQ08 QQ21 QQ41 QRQ QR QR48 QR56 QR40 QR42 QRQQ QR QR QR40 QR40 QR42 QR80 QS16 QS25 QS33 QS34 QS36 QX01 QX02 QX10 4B064 AG27 CA10 CA20 CC01 CC24 DA01 DA13 4B065 AA01X AA58X AA72X AA90X AA93Y AB01 AC14 AC20 BA02 CA24 AAZA ZA52A22 ZA02 CA24 CA44 CA46 4C084 AA02 ZA0 A2 BA22 CA14 CA14 CA46 CAC BB11 CC02 CC04 CC05 DD22 DD23 DD32 DD33 DD41 DD43 EE01 GG01 GG08 4H045 AA10 AA11 A A20 AA30 BA10 BA41 CA40 DA75 DA76 EA20 EA50 FA71 FA72 FA74

Claims (43)

[Claims] 1. An isolated polypeptide, which comprises: a) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 2
A mature form of an amino acid sequence selected from the group consisting of 2; b) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 2
A variant of a mature form of an amino acid sequence selected from the group consisting of 2, wherein any amino acid in the mature form is changed to a different amino acid, provided that 15% of the mature form of the sequence is A variant in which only amino residues are so altered; c) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 2
An amino acid sequence selected from the group consisting of 2; d) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 2
A variant of an amino acid sequence selected from the group consisting of 2, wherein any amino acid specified in the selected sequence is changed to a different amino acid, provided that 15% of the amino acid residues in the sequence remain. A polypeptide comprising an amino acid sequence selected from the group consisting of a variant in which only the group is so modified; and e) a fragment of any of a) to d). 2. The polypeptide of claim 1, wherein the polypeptide is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22. A polypeptide which is a naturally occurring allelic variant of the sequence described. 3. The polypeptide of claim 2, wherein the variant is a translated single nucleotide polymorphism. 4. The polypeptide of claim 1, wherein the polypeptide is the variant polypeptide of claim 1, wherein any amino acid specified in the selected sequence. The polypeptide is modified to provide a conservative substitution. 5. An isolated nucleic acid molecule which comprises: a) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 2
A mature form of the amino acid sequence given in 2; b) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 2
A variant of a mature form of an amino acid sequence selected from the group consisting of 2, wherein any amino acid in the mature form of the selected sequence is changed to a different amino acid, provided that A variant in which only 15% of the amino acid residues in the sequence are so altered; c) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 2
An amino acid sequence selected from the group consisting of 2; d) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 2
A variant of an amino acid sequence selected from the group consisting of 2, wherein any amino acid specified in the selected sequence is changed to a different amino acid, provided that only 15% of the amino acid residues in the sequence are A variant which is not so modified; e) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 2
A nucleic acid fragment encoding at least part of a polypeptide comprising an amino acid sequence selected from the group consisting of 2, or any variant of said polypeptide, wherein any amino acid of said selected sequence Is changed to a different amino acid,
Provided that, however, only 10% of the amino acid residues in said sequence are so altered; and f) encode a polypeptide comprising an amino acid sequence selected from the group consisting of the complement of any of said nucleic acid molecules. A nucleic acid molecule comprising a nucleic acid sequence. 6. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally occurring allelic nucleic acid variant. 7. A nucleic acid molecule according to claim 5 which encodes a variant polypeptide, wherein the variant polypeptide has the polypeptide sequence of a naturally occurring polypeptide variant. . 8. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises a single nucleotide polymorphism encoding the variant polypeptide. 9. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule is: a) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21
A nucleotide sequence selected from the group consisting of: b) a nucleotide sequence, wherein SEQ ID NOs: 1, 3, 5, 7, 9, 11
, 13, 15, 17, 19 or 21 wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of are changed from a nucleotide selected from the group consisting of the selected sequence to a different nucleotide, A nucleotide sequence in which only 15% of the nucleotides are so altered; c) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21.
A nucleic acid fragment of a sequence selected from the group consisting of: and d) a nucleic acid fragment, wherein SEQ ID NOs: 1, 3, 5, 7, 9, 11
, 13, 15, 17, 19 or 21 wherein one or more nucleotides in the nucleotide sequence selected from the group consisting of are changed from a nucleotide selected from the group consisting of the selected sequence to a different nucleotide, A nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: a nucleic acid fragment in which only 15% of the nucleotides are so altered. 10. The nucleic acid molecule according to claim 5, wherein the nucleic acid molecule consists of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21. A nucleic acid molecule that hybridizes under stringent conditions to a nucleotide sequence selected from the group or a complementary strand of the nucleotide sequence. 11. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule is: From a nucleotide selected from the group consisting of:
A nucleotide sequence in which only% nucleotides are so altered, or any fragment thereof, an isolated second complementary to the first polynucleotide.
A polynucleotide of claim 1. 12. A vector comprising the nucleic acid molecule of claim 11. 13. The vector of claim 12, further comprising a promoter operably linked to the nucleic acid molecule. 14. A cell comprising the vector according to claim 12. 15. A polypeptide that immunospecifically binds to the polypeptide according to claim 1,
antibody. 16. The antibody according to claim 15, wherein the antibody is a monoclonal antibody. 17. The antibody of claim 15, wherein the antibody is a humanized antibody. 18. A method for determining the presence or amount of the polypeptide of claim 1 in a sample, the method comprising: (a) providing the sample; (b) Introducing a sample into an antibody that immunospecifically binds to the polypeptide; and (c) determining the presence or amount of the antibody that binds to the polypeptide, and thereby the presence or amount of the polypeptide in the sample. A method comprising the step of measuring 19. A method for determining the presence or amount of the nucleic acid molecule of claim 5 in a sample, the method comprising: (a) providing the sample; (b) the Introducing a sample into a probe that binds to the nucleic acid molecule; and (c) determining the presence or amount of the probe that binds to the nucleic acid molecule, thereby determining the presence or amount of the nucleic acid molecule in the sample. A method comprising the step of: 20. A method of identifying a factor that binds to the polypeptide of claim 1, comprising: (a) introducing the polypeptide into the factor; and (b) A method comprising determining whether an agent binds to the polypeptide. 21. A method for identifying potential therapeutic agents for use in the treatment of a condition wherein said condition is aberrant expression or aberration of the polypeptide of claim 1. In relation to physiological interactions, the method comprises: (a) providing a cell which expresses the polypeptide of claim 1 and which has properties or functions attributable to said polypeptide; (b) said Contacting the cells with a composition containing the candidate substance; and (c) determining whether the substance alters the predisposition or the function attributable to the polypeptide, thereby the presence of the substance. A method comprising the step of identifying a substance as a potential therapeutic agent if the alterations observed below are not observed when the cells are contacted with a composition lacking the substance. 22. A method for modulating the activity of the polypeptide of claim 1, wherein the method comprises treating a sample of cells expressing the polypeptide of claim 1 with the activity of the polypeptide. A method comprising introducing with a compound that binds to the polypeptide in an amount sufficient to modulate. 23. A method of treating or preventing a condition associated with the polypeptide of claim 1, wherein the method comprises administering to a subject for whom such treatment or prevention is desired. A method comprising administering the polypeptide of claim 1 in an amount sufficient to treat or prevent the condition. 24. The method of claim 23, wherein the subject is a human. 25. A method of treating or preventing a condition associated with the polypeptide of claim 1, wherein said method is to a subject for whom such treatment or prevention is desired. A method comprising administering a NOVX nucleic acid in an amount sufficient to treat or prevent the condition. 26. The method of claim 25, wherein the subject is a human. 27. A method of treating or preventing a condition associated with the polypeptide of claim 1, wherein the method comprises administering to a subject for whom such treatment or prevention is desired. A method comprising administering a NOVX antibody in an amount sufficient to treat or prevent the condition. 28. The method of claim 27, wherein the subject is a human. 29. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier. 30. A pharmaceutical composition comprising the nucleic acid molecule of claim 5 and a pharmaceutically acceptable carrier. 31. A pharmaceutical composition comprising the antibody of claim 15 and a pharmaceutically acceptable carrier. 32. A kit comprising the pharmaceutical composition of claim 29 in one or more containers. 33. A kit comprising the pharmaceutical composition of claim 30 in one or more containers. 34. A kit comprising the pharmaceutical composition of claim 31 in one or more containers. 35. Use of a therapeutic agent in the manufacture of a medicament for treating a syndrome associated with a human disease, wherein the disease is selected from the pathologies associated with the polypeptide of claim 1, wherein Use, wherein said therapeutic agent is the polypeptide of claim 1. 36. Use of a therapeutic agent in the manufacture of a medicament for treating a syndrome associated with a human disease, wherein the disease is selected from the pathologies associated with the polypeptide of claim 1, wherein , The therapeutic agent is a NOVX nucleic acid. 37. Use of a therapeutic agent in the manufacture of a medicament for treating a syndrome associated with a human disease, wherein the disease is selected from the pathologies associated with the polypeptide of claim 1, wherein , The therapeutic agent is a NOVX antibody. 38. A method for screening a modulator of activity or latency for a pathological condition associated with the polypeptide of claim 1 or a predisposition for said pathological condition, which method comprises the following: a) Administering a test compound to a test animal of increased risk for a condition associated with the described polypeptide, wherein the test animal is
Recombinantly expressing the polypeptide according to claim 1; b) measuring the activity of said polypeptide in said test animal after the step of administering said compound of step (a); and c) said in said test animal. Comparing the activity of the protein to the activity of the polypeptide in a control animal to which the polypeptide has not been administered, wherein the change in the activity of the polypeptide in the test animal compared to the control animal Comprising the step of indicating that said test compound is a modulator of latency of a disease state associated with the polypeptide of claim 1 or a predisposition to said disease state. 39. The method of claim 38, wherein the test animal expresses the test protein transgene or is under the control of a promoter at an increased level as compared to a wild-type test animal. A method of recombinant test animals expressing the transgene in, wherein the promoter is not the native gene promoter of the transgene. 40. A method for determining the presence or predisposition to a disease associated with altered levels of the polypeptide of claim 1 in a first mammalian subject, the method comprising: The following: a) measuring the expression level of the polypeptide in a sample from the first mammalian subject; and b) the amount of the polypeptide in the sample of step (a) Comparing the amount of the polypeptide present in a control sample from a second mammalian subject, known to be absent or known to be less susceptible to the disease, wherein: , A change in the expression level of the peptide in the first subject as compared to the control sample is indicative of the presence of or predisposition to the disease. 41. A method for determining the presence or predisposition to a disease associated with altered levels of the nucleic acid molecule of claim 5 in a first mammalian subject, said method comprising: The following: a) measuring the amount of the nucleic acid in a sample from the first mammalian subject;
And b) the amount of said nucleic acid in said sample of step (a) is from a second mammalian subject known to be free of or susceptible to said disease Comparing the amount of said nucleic acid present in a control sample, wherein the change in the level of said nucleic acid in said first subject as compared to said control sample is due to the presence of said disease or said A method comprising the step of indicating a predisposition to a disease. 42. A method of treating a pathological condition in a mammal comprising:
The method comprises the step of administering to the mammal a polypeptide in an amount sufficient to ameliorate the pathological condition, wherein the polypeptide is SEQ ID NO: 2, 4, 6, 8, 10.
, A polypeptide having an amino acid sequence at least 95% identical to a polypeptide comprising an amino acid sequence selected from the group consisting of 12, 14, 16, 18, 20 or 22 or a biologically active fragment thereof. Method. 43. A method of treating a pathological condition in a mammal comprising:
16. The method, wherein the method comprises the step of administering to the mammal the antibody of claim 15 in an amount sufficient to ameliorate the pathological condition.