JP2002537811A - Leukocytes and blood-related proteins - Google Patents

Abstract

  (57) [Summary] The present invention provides proteins associated with human leukocytes and blood (LBAP) and polynucleotides that identify and encode LBAP. The present invention also provides expression vectors, host cells, antibodies, agonists and antagonists. Furthermore, the present invention provides a method for diagnosing, treating or preventing a disease associated with the expression of LBAP.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field The present invention relates to nucleic acid sequence and amino acid sequence of a protein associated with white blood cells and blood, as well as the diagnosis of cell proliferative disorders, including autoimmune disorders / inflammatory diseases as well as cancer, these sequences in the treatment and prevention It is about usage.

BACKGROUND OF THE INVENTION The blood circulatory system is composed of various cell types, including white blood cells, red blood cells and plasma. Leukocytes are part of the immune system and are responsible for the immune response. A particularly important function of the immune response is antigen recognition. Some leukocytes are activated by a cellular immune response and react with foreign antigens on the surface of other host cells. These antigens are carried by the blood, and levels of these antigens may indicate the progression of a particular disease or disorder. For example, in some cancers, plasma may carry cell surface proteins such as tumor cell markers. Because of these and other interactions, particularly important information in the diagnosis, treatment and prevention of various diseases and disorders can be obtained by understanding the properties and functions of leukocytes and blood-associated proteins.

[0003] Circulation of cell surface proteins associated with the circulatory system includes cell signaling receptors and tumor antigens. One of the tumor antigens found in serum is carcinoembryonic antigen (
CEA). CEA is a colon, pancreas, lung, breast and well-established glycoprotein marker adenocarcinoma ovary (Cotran, R. et al (1994 ), Robbins Pathologica l Basis of Disease 5 th edition, WB Saunders Company , Philadelphia PA,
pp. 300-301). CEA is a CA that is regularly monitored in the serum of cancer patients
It belongs to a small group of protein molecules similar to muclin, prostate specific antigen and α-fetoprotein, such as -125 (Cotran, sunra).

[0004] Fc receptors, interleukin-2 receptors, and lymphokine-activated killers (LA
K) Leukocyte membrane proteins such as other lymphokine receptors in cells are important cell membrane proteins involved in the immune response to tumors (Brostoff, J. et.
al. (1991) Clinical Immunology , Mosby, London, England, pp.20.8-20.12)
. The V7 gene product, another membrane surface protein found in LAK cells, plays a role in T cell activation. V7, a cell surface glycoprotein on antigen-activated T cells, monocytes and granulocytes, has an extracellular domain similar to an immunoglobulin (Ig) -like domain containing seven pairs of cysteine residues. V7 may be a new member of the Ig gene superfamily and has a functional role in the immune response (Ruegg, C. et al. (1995) J. Immunol. 35 154: 44434-44443).

[0005] Cell surface membrane-bound microbial superantigens have a role in the pathogenesis of autoimmune disorders. An example is the human endogenous retrovirus (HERV) superantigen. By specialized antigen presenting cells such as macrophages and dendritic cells
Cell surface expression of HERV-derived superantigen induces β-cell destruction in the pancreas by systemic activation of self-reactive T cells (Conrad, B. et al. (1991) Cel
l 90: 303-313).

[0006] Another class of circulatory related cell surface molecules are the receptors involved in hematopoiesis. Binding of hematopoietic growth factors and cytokines to specific cell surface receptor proteins triggers intracellular signaling, resulting in proliferation and differentiation of blood cell precursors. These cell surface receptor proteins include interleukin-2α and interleukin-2β, erythropoietin and various colony stimulating factors. Each of these receptors is a transmembrane protein with an extracellular ligand binding domain that is activated by direct interaction with a cognate ligand (Kaczma
rski, R. and G. Jufti (1991) Blood Rev. 5: 193-203).

[0007] Disclosure of novel leukocyte and blood-related proteins and polynucleotides encoding them provides new compositions useful for the diagnosis, prevention and treatment of autoimmune disorders / inflammatory diseases and cell proliferative disorders including cancer To meet the needs of those skilled in the art.

SUMMARY OF THE INVENTION [0008] The present invention collectively refers to "LBAP", "LBAP-1", "LBAP-2", "LBAP-3", "LBAP-
It is characterized by leukocytes and blood-related proteins, which are purified polypeptides designated "4" and "LBAP-5". In one embodiment, the present invention relates to (a) SEQ ID NO: 1-5
An amino acid sequence selected from the group consisting of: (b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5; (c) A biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, or (d) an immunogenicity of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5 An isolated polypeptide comprising a fragment is provided.

Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO: 1-5
A biologically active fragment of an amino acid sequence selected from the group consisting of: or (d) SE
Provided is an isolated polynucleotide encoding a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of Q ID NOs: 1-5. Alternatively, the polynucleotide is selected from the group consisting of SEQ ID NO: 6-10.

Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO: 1-5
A biologically active fragment of an amino acid sequence selected from the group consisting of: or (d) SE
A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of Q ID NO: 1-5 . Alternatively, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a genetic transformant comprising the recombinant polynucleotide.

The present invention also relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO: 1-5
A biologically active fragment of an amino acid sequence selected from the group consisting of: or (d) SE
Provided is a method for producing a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of Q ID NOs: 1-5. The method includes (a) culturing cells under conditions suitable for expression of a polypeptide, the cells comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide. Culturing steps such as transformation with the recombinant polynucleotide, and (b) recovering the polypeptide so expressed.

Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO: 1-5
A biologically active fragment of an amino acid sequence selected from the group consisting of: or (d) SE
An isolated antibody that specifically binds to a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of Q ID NOs: 1-5.

Further, the present invention provides (a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 6-10, and (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 6-10. A naturally occurring polynucleotide sequence having at least 70% sequence identity to (c) a polynucleotide sequence complementary to (a), or (d) (b)
Provided an isolated polynucleotide comprising a polynucleotide sequence complementary to Alternatively, the polynucleotide comprises at least 60 contiguous nucleotides.

Further, the present invention provides (a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 6-10, and (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 6-10. A naturally occurring polynucleotide sequence having at least 70% sequence identity to (c) a polynucleotide sequence complementary to (a), or (d) (b)
Comprising a polynucleotide sequence comprising a polynucleotide sequence complementary to
Methods are provided for detecting a target polynucleotide in a sample. The method includes the steps of (a) hybridizing a sample to a probe comprising at least 16 contiguous nucleotides comprising a sequence complementary to a target polynucleotide in the sample, wherein the probe and the target A hybridization process in which the probe specifically hybridizes to the target polynucleotide under conditions where a hybridization complex is formed between the polynucleotides; and (b) whether the hybridization complex exists This includes a step of detecting whether or not the amount is present, and, if present, optionally detecting the amount thereof. Alternatively,
The probe contains at least 30 contiguous nucleotides. Alternatively, the probe comprises at least 60 contiguous nucleotides.

Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO: 1-5
A biologically active fragment of an amino acid sequence selected from the group consisting of: or (d) SE
Provided is a pharmaceutical composition comprising an effective amount of a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of Q ID NO: 1-5, and a pharmaceutically acceptable excipient. . Further, the present invention provides a method of treating a disease or condition associated with reduced expression of functional LBAP, comprising administering the pharmaceutical composition to a patient in need of such treatment. I do.

Further, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO: 1-5
A biologically active fragment of an amino acid sequence selected from the group consisting of: or (d) SE
Provided is a method for screening a compound comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of Q ID NOs: 1-5 for the effectiveness as an agonist of a polypeptide. The method includes (a) exposing a sample containing the polypeptide to the compound, and (b) detecting the activity of the agonist in the sample. Alternatively, the present invention provides a pharmaceutical composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Alternatively, the present invention provides a method of treating a disease or condition associated with reduced expression of functional LBAP, the method comprising administering the pharmaceutical composition to a patient in need of such treatment. I do.

Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO: 1-5
A biologically active fragment of an amino acid sequence selected from the group consisting of: or (d) SE
Provided are methods for screening compounds for the effectiveness as antagonists of a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of Q ID NOs: 1-5. The method includes (a) exposing a sample containing the polypeptide to the compound, and (b) detecting the activity of the antagonist in the sample. Alternatively, the invention provides a pharmaceutical composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Alternatively, the invention provides a method of treating a disease or condition associated with overexpression of functional LBAP, comprising administering the pharmaceutical composition to a patient in need of such treatment. .

Further, the present invention provides a method for screening a compound for efficacy in altering the expression of a target polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 6-10, wherein the method is provided. The methods include (a) exposing a sample containing the target polypeptide to the compound, and (b) detecting a change in expression of the target polypeptide.

[0019] Proteins form the present invention for carrying out the invention, nucleic acid sequences, and before describing how the present invention,
It should be understood that the invention is not limited to the particular devices, materials, and methods disclosed herein, but that embodiments thereof can be varied. The terminology used herein is used for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Please also understand.

In this specification, including the claims, the singular forms “a” and “the”
Note that the notation also has multiple meanings unless the context clearly indicates otherwise. Thus, for example, reference to "a host cell" includes a plurality of such host cells, and reference to "an antibody" includes reference to one or more antibodies and their equivalents well known to those of skill in the art. Represents.

Unless defined otherwise, all technical and specific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials are described herein. All references mentioned herein are cited to describe and disclose the cell lines, protocols, reagents and vectors that can be used in connection with the present invention as reported in the literature. Nothing in this disclosure should be construed as an admission that the present invention cannot precede such disclosure by the prior invention.

The definition term "LBAP" is obtained from any species, particularly mammalian species, including cows, sheep, pigs, mice, horses, and humans, and may be natural, synthetic, semi-synthetic, or Refers to the amino acid sequence of substantially purified LBAP obtained from any source, such as recombinant.

The term “agonist” refers to a molecule that enhances or mimics the biological activity of LBAP. The agonists include proteins, nucleic acids, carbohydrates, small molecules or any other compounds or compositions that interact directly with LBAP or act on components of the biological pathways involving LBAP. Regulates the activity of LBAP.

The term “allelic variant” refers to another form of the gene encoding LBAP. An allelic variant is a mutated mRNA resulting from at least one mutation in a nucleic acid sequence.
Alternatively, polypeptides may form, but their structure or function may or may not change. Some genes do not have a naturally occurring allelic variant, some have one, or many exist. Common mutations that generally result in allelic variants are due to natural deletions, additions or substitutions of nucleotides. Each of these forms of change, alone or in combination with other changes, can occur one or more times in a given sequence.

As used herein, an “altered” nucleic acid sequence encoding LBAP includes various nucleotides that result in a polynucleotide encoding the same LBAP or a polypeptide comprising at least one of the functional characteristics of LBAP. And those sequences with deletions, insertions or substitutions of This definition includes improper or unexpected hybridization of an LBAP-encoding polynucleotide sequence to an allelic variant at a position different from the normal chromosomal location, or specific oligonucleotide probes of the LBAP-encoding polynucleotide. It includes polymorphisms that are easily detectable or difficult to detect. The encoded protein may also be "mutated", resulting in a silent change resulting in a functionally equivalent LBAP
Substitutions, deletions or insertions of the following amino acid residues. Intentional amino acid substitutions may result in similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues, as long as the biological or immunological activity of LBAP is retained. Can be performed based on For example, negatively charged amino acids include aspartic acid and glutamic acid, and positively charged amino acids include lysine and arginine.
Amino acids having uncharged polar side chains with similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; and phenylalanine and tyrosine.

The term “amino acid” or “amino acid sequence” is an oligopeptide, peptide, polypeptide or protein sequence or any fragment thereof and refers to a natural or synthetic molecule. When "amino acid sequence" is used to refer to the amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and similar terms refer to the complete, intact amino acid sequence associated with the described protein molecule. It is not limited to an array.

[0027] The term "amplification" refers to the production of additional copies of a nucleic acid sequence, usually performed using the polymerase chain reaction (PCR) technique well known to those skilled in the art.

The term “antagonist” is a molecule that inhibits or attenuates the biological activity of LBAP. Antagonists include proteins, such as antibodies, nucleic acids, carbohydrates, small molecules or any other compounds or compositions, which interact directly with LBAP, or
Modulates the activity of LBAP by interacting with components of the biological pathways involving LBAP.

The term “antibody” refers to intact molecules, Fa, F (ab ′) ₂ , and Fv that can bind antigenic determinants.
Refers to those fragments, such as fragments. Antibodies that bind LBAP polypeptides can be prepared using untreated polypeptides or using fragments thereof containing small peptides that participate as antigens to be immunized. The polypeptide or oligopeptide used to immunize an animal (eg, a mouse, rat, or rabbit) is obtained from translated or chemically synthesized RNA and can be coupled to a carrier protein if necessary. Commonly used carriers that chemically bind to peptides include:
Includes bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin (KLH). The animal is then immunized with the conjugated peptide.

The term “antigenic determinant” refers to a region of a molecule (ie, an epitope) that makes contact with a particular antibody. When a host animal is immunized with a protein or protein fragment, many regions of the protein trigger the production of antibodies that specifically bind to antigenic determinants (specific regions or three-dimensional structures of the protein). I can do it. The antigenic determinant is
It can compete with the original antigen (ie, the immunogen used to elicit the immune response) for binding to the antibody.

The term “antisense” refers to any composition that can base pair with the “sense” strand of a particular nucleic acid sequence. Antisense compositions include DNA; RNA; peptide nucleic acid (PN
A); an oligonucleotide having a modified backbone linkage such as phosphorothionate, methylphosphonate or benzylphosphonate; having a modified sugar such as 2'-methoxyethyl sugar or 2'-methoxyethoxy sugar Oligonucleotides; or 5-methylcytosine, 2'-deoxyuracil or 7
Oligonucleotides with modified bases such as -deaza-2'-deoxyguanosine may be included. Antisense molecules can be produced by any method, including chemical synthesis and transcription. Once introduced into a cell, a complementary antisense molecule base pairs with the native sequence produced by the cell to form a duplex, preventing transcription and translation. The expression “negative” or “minus (−)” may refer to the antisense strand of the reference DNA molecule, and the expression “positive” or “plus (+)” may refer to the sense strand.

The term “biologically active” refers to a protein that has a structural, regulatory or biochemical function of a naturally occurring molecule. Similarly, "immunologically active" refers to natural LBAP that elicits a specific immune response in the appropriate animal or cell and binds to a specific antibody.
, Recombinant LBAP or synthetic LBAP or any oligopeptide thereof.

The terms “complementary” or “complementarity” refer to the natural joining of polynucleotides by base pairing. For example, the sequence "5'AG-T3 '" binds to the complementary sequence "3'TC-A5'". Complementarity between two single-stranded molecules can be either “partial”, such as when only a few nucleic acids are bound, or “complete”, such as when there is complete complementarity between the single-stranded molecules. Some are. The degree of complementarity between nucleic acid strands greatly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions that depend on the binding of nucleic acid strands and in the design and use of peptide nucleic acid (PNA) molecules.

The terms “composition comprising a given polynucleotide sequence” and “composition comprising a given amino acid sequence” generally refer to any composition comprising a given polynucleotide or amino acid sequence. The composition may include a dry formulation or an aqueous solution. A composition containing a polynucleotide encoding LBAP or a fragment of LBAP can be used as a hybridization probe. The probe can be stored in a lyophilized state and can be conjugated to a stabilizing agent such as a carbohydrate. In hybridization, the probe is placed in an aqueous solution containing a salt (eg, NaCl), a surfactant (eg, sodium dodecyl sulfate (SDS)) and other substances (eg, Denhardt's solution, dried milk, salmon sperm DNA, etc.). Can be dispersed.

The term “consensus sequence” refers to a nucleic acid sequence obtained by resequencing and separating unnecessary nucleotides, or using a XL-PCR kit (The Perkin Elmer Corp., Norwalk, Conn.) In the 5 ′ direction and / or 3 ′ direction. A nucleic acid sequence that has been extended and resequenced in the 'direction, or a computer program for assembling fragments (eg, GELVIEW Fragment Assembly system,
GCG, Madison WI) is a nucleic acid sequence assembled from two or more insight clones and, optionally, one or more overlapping sequences of one or more proprietary ESTs. Some sequences are extended and combined to create a consensus sequence.

The term “conservative amino acid substitution” refers to a substitution that substantially impairs the properties of the original protein, that is, the substitution conserved without significantly altering the structure or function of the protein. . The following shows conservative amino acid substitutions where the original amino acid of the protein is replaced with another amino acid. Original residue Conservative substitution Ala Gly, Set Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln , Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile , Leu, Thr In general, in conservative amino acid substitution, (a) the main chain structure of the polypeptide in the substitution region, for example, β-sheet or α-helix conformation, (b) the charge or hydrophobicity of the molecule at the substitution site and And / or (c) most of the side chains are retained.

The term “deletion” refers to a change in the nucleotide or amino acid sequence that results in the deletion of one or more nucleotide or amino acid residues.

The term “derivative” refers to a chemically modified polypeptide or polynucleotide sequence. Chemical modification of a polynucleotide sequence can include, for example, replacement of hydrogen with an alkyl, acyl, hydroxyl, or amino group. A polynucleotide derivative encodes a polypeptide that retains the biological or immunological functions of the native molecule. Polypeptide derivatives have been modified by glycosylation, polyethylene glycolation (pegylation), or any other process that preserves the biological or immunological function of the original polypeptide.

The term “fragment” refers to a unique portion of LBAP or a polynucleotide encoding LBAP that is identical to, but shorter in length than, its parent sequence. "Fragments" can include those of a given sequence length minus one nucleotide / amino acid residue. For example, for some fragments, 5-1000
Consecutive nucleotide or amino acid residues may be included. Probes, primers, antigens, therapeutic molecules or fragments used for other purposes are at least 5, 10, 1
It can be 5, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from particular regions of the molecule. For example, a polypeptide fragment may contain the first 250 or 500 amino acids as shown in the given sequence (or the first 25 or 25 amino acids of the polypeptide).
% Or 50%). These lengths are exemplary, and examples of the invention may include any of the lengths described in the specification, including the sequence listings, tables and figures.

Certain fragments of SEQ ID NO: 6-10 include, for example, regions of a unique polynucleotide sequence that specifically identify SEQ ID NO: 6-10, which are different from other sequences in the same genome. Is included. Certain fragments of SEQ ID NO: 6-10 are useful, for example, in hybridization and amplification techniques, and similar methods that distinguish SEQ ID NO: 6-10 from related polynucleotide sequences. A fragment of SEQ ID NO: 6-10 that matches a fragment or SEQ ID NO: 6-10
The exact length of the region can be routinely determined by known techniques based on the purpose of the fragment.

A fragment of SEQ ID NO: 1-5 is encoded by a fragment of SEQ ID NO: 6-10. Certain fragments of SEQ ID NO: 1-5 include regions of a unique amino acid sequence that specifically identify SEQ ID NO: 1-5. For example, one fragment of SEQ ID NO: 1-5
: Useful as an immunogenic peptide for the production of an antibody that specifically recognizes 1-5. The exact length of a fragment of SEQ ID NO: 1-5 or a region of SEQ ID NO: 1-5 that matches a fragment can be routinely determined by well-known techniques based on the purpose of the fragment.

The term “similarity” refers to a degree of complementarity. Similarity includes partial similarity and complete similarity. “Identity” may be used instead of the term “similarity”.
Partially complementary sequences that at least partially inhibit the same sequence from hybridizing to the target nucleic acid are referred to as "substantially similar." Inhibition of hybridization between a completely complementary sequence and the target sequence can be assayed under mild stringency conditions using a hybridization assay (such as Southern blot, Northern blot or solution hybridization). Substantially similar sequences or hybridization probes can compete for and inhibit the binding of perfectly similar (identical) sequences to the target sequence under mildly stringent conditions.
This does not mean that non-specific binding is tolerated under mildly stringent conditions, and under mildly stringent conditions, the mutual binding of the two sequences is specific (ie, (Selective) interaction. The absence of non-specific binding does not even have partial complementarity (
That is, less than about 30% similarity or identity) can be tested by using a second target sequence. In the absence of non-specific binding, a substantially similar sequence or probe will not hybridize to a second non-complementary target sequence.

The terms “percent identity” or “percent identity” for a polynucleotide sequence refer to the identity of residue matches between at least two or more polynucleotide sequences aligned using standard algorithms. Means percentage. Such algorithms use gaps in the compared sequences to make a more significant comparison between the two sequences in a standard and reproducible way to optimize the alignment between the two sequences be able to.

The percent identity between polynucleotide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated in the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package and includes a suite of molecular biology analysis programs (DNASTAR, Madison
WI). This CLUSTAL V is described in Higgins, DG and PM Sharp (198
9) CABIOS 5: 151-153 and Higgins, DG et al. (1992) CABIOS 8: 189-191. For alignment of pairs of polynucleotide sequences, the default parameters are set as Ktuple = 2, gap penalty = 5, window = 4 and “diagonals saved” = 4. The "weighted" residue weight table is selected as the default.
CLUSTAL V reports the percent identity as the "percent similarity" between a pair of aligned polynucleotide sequences.

Alternatively, a commonly used set of freely available sequence comparison algorithms is:
NCBI, Bethesda, MD, and the Internet (http: // www.ncbi.nlm.nih.gov/BLAS T /) National Center available from several sources, including such as for Biotechn
ology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Alt
schul, SF et al. (1990) J. Mol. Biol. 215: 403-410). BLA
The ST software suite includes a variety of sequence analysis programs, including "blastn", used to align known polynucleotide sequences with other polynucleotide sequences from various databases. A tool called "BLAST 2 Sequences" is available and is used to directly compare pairs of two nucleotide sequences. "BLAST 2 Sequences" is available interactively by accessing http://www.ncbi.nlm.nih.gov/gorf/b12.html . The “BLAST 2 Sequences” tool uses b
It can be used for both lastn and blastp (described below). The BLAST program is
Generally used with gaps and other parameters set to default settings. For example, when comparing two nucleotide sequences, the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-199) set to default parameters
9) can be used with blastn. Such default parameters are, for example,
It can be as follows. Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: −2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off: 50 Expect: 10 Word Size: 11 Filter: on Or a fragment obtained from a shorter sequence, such as a fragment obtained from a larger predetermined sequence (eg, at least 20, 30, 40, 50, 70). , 100 or 200 contiguous nucleotide fragments). Such lengths are merely exemplary, and the length at which percent identity can be determined using fragments of any length of the sequences set forth in the specification, including the sequence listing, tables and figures. Should be understood.

In some cases, similar amino acid sequences are encoded due to the degeneracy of the genetic code, despite nucleic acid sequences that do not show high identity. Nucleic acid sequences can be varied using degeneracy to create multiple nucleic acid sequences such that they all encode substantially the same protein.

The term “percent identity” or “percent identity” for a polypeptide sequence
By means the percentage of residue matches between at least two or more polypeptide sequences aligned using standard algorithms. Methods for polypeptide sequence alignment are well known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, as detailed above, usually preserve the acidity and hydrophobicity of the substitution site and preserve the structure (and thus function) of the polypeptide.

The percent identity between polypeptide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated in the MEGALIGN version 3.12e sequence alignment program (described above). For alignment of pairs of polypeptide sequences using CLUSTAL V, the default parameters are Ktuple = 1, gap
Set penalty = 3, window = 5 and “diagonals saved” = 5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity between pairs of aligned polypeptide sequences is reported by CLUSTAL V as "percent similarity".

Alternatively, the NCBI BLAST software suite can be used. For example, when comparing pairs of two polypeptide sequences, "BLAS" set with default parameters is used.
The T2 Sequences tool version 2.0.9 (May-07-1999) can be used with blastp. Such default parameters can be, for example, as follows. Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: 1 penalties Gap x drop-off: 50 Expect: 10 Word Size: 3 Filter: on Or fragments obtained from shorter sequences, eg, larger predetermined polypeptide sequences (eg, at least 15, 20, 30,
(A fragment of 40, 50, 70 or 150 contiguous residues).
Such lengths are merely exemplary, and the length at which percent identity can be determined using fragments of any length of the sequences set forth in the specification, including the sequence listing, tables and figures. Should be understood.

“Human artificial chromosomes” (HACs) are linear small chromosomes that contain a DNA sequence between 6 kb and 10 Mb in size and that contain all the elements necessary for stable mitotic chromosome isolation and maintenance. is there.

The term “humanized antibody” refers to an antibody molecule in which the amino acid sequence in the non-antibody binding region has been changed so that the antibody is closer to a human antibody while retaining its original binding ability.

The term “hybridization” refers to an annealing process by which a single-stranded polynucleotide base pairs with a complementary strand under defined hybridization conditions. Specific hybridization means that two nucleic acid sequences have a high degree of identity. Specific hybridization complexes are formed under conditions that permit annealing, and remain hybridized after the "washing process." The washing step is particularly important in determining the stringency of the hybridization process; under more stringent conditions, non-specific binding (ie, pair binding between nucleic acid strands that are not perfectly matched) is reduced. The conditions under which annealing between nucleic acid sequences is permissible are routinely determined by those skilled in the art and are constant during a hybridization experiment, while washing conditions can be varied during the experiment for the desired stringency Thus, hybridization specificity is obtained. Conditions that allow annealing include, for example, at about 68 ° C., about 6 × SSC, about 1% (w / v) SDS, and about 100 μg / ml denatured salmon sperm DNA.
Holds in the presence of

In general, some of the stringency of hybridization can be represented by the temperature at which the washing step is performed. Typically, this washing temperature is selected to be about 5-20 ° C. below the melting point (Tm) of the particular sequence at a given ionic strength and pH. This Tm is (
The temperature at which 50% of the target sequence (under a given ionic strength and pH) hybridizes with a perfectly matched probe. The formula for calculating Tm and the conditions for nucleic acid hybridization are well known, and are described in Sambrook et al. (1989, Molecular Cloning : A Laboratory Manual , 2nd edition, volume 1-3, Cold Spring Harbor Press, Plainview
NY; see especially Volume 2, Chapter 9).

High stringency conditions for hybridization between polynucleotides of the present invention include a 1 hour washing step at about 68 ° C. in the presence of about 0.2 × SSC and about 1% SDS. Alternatively, it is performed at a temperature of 65 ° C, 60 ° C, 55 ° C or 42 ° C. The concentration of SSC can vary from about 0.1 to 2 × SSC in the presence of about 0.1% SDS. Usually, blocking agents are used to block non-specific hybridization. Such blocking agents include, for example, denatured salmon sperm DNA at about 100-200 μg / ml. Organic solvents such as formamide at a concentration of about 35-50% v / v can be used in certain cases, such as, for example, hybridization of RNA and DNA. Useful alterations of these washing conditions will be readily apparent to those skilled in the art. Hybridization under particularly high stringency conditions may indicate evolutionary similarity between the nucleotides.
Such similarities strongly suggest a similar role for their nucleotides and encoded polypeptides.

The term “hybridization complex” refers to a complex formed between two nucleic acid sequences by the force of hydrogen bond formation between complementary bases. A hybridization complex may either be formed in solution (e.g., C ₀ t or R ₀ t analysis), or one of the nucleic acids present in solution and the solid support (e.g., paper, membranes, filters,
A pin or glass slide, or other suitable nucleic acid immobilized on a cell or other suitable substrate on which the nucleic acid is immobilized.

The term “insertion” or “addition” refers to a change in a nucleotide or amino acid sequence that adds one or more nucleotides or amino acid residues, respectively.

The term “immune response” refers to a condition associated with inflammation, trauma, immune disorders or infectious or inherited diseases. These conditions are characterized by the expression of various factors that affect cells and the systemic defense system, such as cytokines, chemokines and other signaling molecules.

The term “immunogenic fragment” is a polypeptide or oligopeptide fragment of LBAP that can elicit an immune response when introduced into an organism (eg, a mammal). Also,
The term "immunogenic fragment" includes all polypeptide or oligopeptide fragments of LBAP that are useful in any of the antibody production methods disclosed or known in the art.

The term “microarray” refers to an arrangement of various polynucleotides on a substrate.

The term “element” or “array element” in the microarray context means
Refers to a hybridizable polynucleotide located on the surface of a substrate.

The term “modulate” refers to a change in the activity of LBAP. For example, modulation results in an increase or decrease in protein activity, binding properties, or any other biological, functional, or immunological properties of LBAP.

The term “nucleic acid” or “nucleic acid sequence” refers to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These terms also refer to single-stranded or double-stranded, sense or antisense strands of genomic or synthetic origin.
Alternatively, it refers to RNA, peptide nucleic acid (PNA), or any DNA-like or RNA-like substance.

The term “operably binds” refers to a state in which a first nucleic acid sequence and a second nucleic acid sequence are in a functional relationship. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. In general, operably linked DNA sequences can be very close together or contiguous if the regions encoding the two proteins need to be joined in the same reading frame.

The term “peptide nucleic acid” (PNA) refers to an antisense molecule or an anti-genetic agent (anti-gene) comprising an oligonucleotide at least 5 or more nucleotides in length attached to a peptide backbone of lysine-terminated amino acid residues. gene agent). Terminal lysine confers solubility to its composition. PNAs preferentially bind to complementary single-stranded DNA or RNA and stop transcript elongation, and can also PNA-polyethylene glycol extend the life of PNAs in cells.

“Probe” refers to a nucleic acid sequence encoding LBAP, its complementary sequence or a fragment thereof, which is used to detect the same nucleic acid sequence, allelic nucleic acid sequence or related nucleic acid sequence. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioisotopes, ligands, chemiluminescent reagents and enzymes. “Primers” are short nucleic acids (usually DNA oligonucleotides) that can anneal to a target polynucleotide by forming complementary base pairs. The primer can then be extended along the target DNA strand by a DNA polymerase enzyme. The primer set can be used, for example, for amplification (and identification) of a nucleic acid sequence by a PCR method.

The probes and primers used in the present invention usually have at least 15
Of consecutive nucleotides. To increase specificity, longer probes and primers can be used, including, for example, at least 20, 25, 30, 40, 50, 60 contiguous sequences of the disclosed nucleic acid sequences. , 70
, 80, 90, 100 or 150 nucleotides. Probes and primers
It should be understood that they may be considerably longer than these examples, and that any length of nucleotides provided herein, including tables, figures and sequence listings, may be used.

For the preparation and use of probes and primers, see, for example, Sambrook,
J. et al., 1989, entitled Molecular Cloning: A Laboratory Manual , 2
Volume 1-3 of the edition (Cold Spring Harbor Press, Plainview NY) or Ausubel, FM
1987, entitled " Current Protocols in Molecular Biology " (Greene
Pubi. Assoc. & Wiley-Intersciences, New York NY) and Innis et al., 1990, entitled " PCR Protocols, A Guide to Methods and Applications " (Ac
ademic Press, San Diego CA). A primer set for PCR is, for example, Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Rese
arch, Cambridge MA) can be obtained from the known sequence using a computer program for that purpose.

Oligonucleotides used as primers are selected by computer programs for selecting primers well known in the art. For example, OLIGO 4.06 software allows the selection of primer pairs for PCR up to 100 nucleotides each,
Useful for analysis of oligonucleotides up to 5,000 nucleotides and larger polynucleotides from input polynucleotide sequences up to the kilobase. Similar primer selection programs incorporate additional features to enhance capacity. For example, PrimOU primer selection program (Genome Center at Unive
rsity of Texas South West Medical Center, available from Dallas TX), which allows you to select specific primers from megabase sequences and
(genome-wide scope). Primer3 primer selection program (Whitehead Institute / MIT Center for Genome Research, Ca
(available from mbridge MA1) allows the user to specify a sequence to be avoided as a primer binding site.
libaray) ". Primer3 is also particularly useful for selecting oligonucleotides for microarrays. (The source code for the latter two primer selection programs can be obtained from their respective sources to meet the specific needs of the user. Can be changed to). PrimerGen program (UK Human
Genome Mapping Project Resource Centre, available from Cambridge UK)
To design primers based on multiple sequence alignments, primers can be selected that hybridize to either the most conserved or the least conserved regions of the aligned nucleic acid sequences. Thus, this program is useful for identifying unique conserved oligonucleotide and polynucleotide fragments. Oligonucleotide and polynucleotide fragments identified by any of the selection methods described above, for example, identify polynucleotides that are completely or partially complementary in PCR or sequencing primers, microarray elements, or nucleic acid samples. It is useful in hybridization technology as a specific probe. The method for selecting the oligonucleotide is not limited to the method described above.

The term “recombinant nucleic acid” is not a naturally-occurring sequence, but a sequence that is an artificial combination of two or more different, spaced apart segments of sequence. This artificial combination
Although it is often carried out by chemical synthesis, more generally, for example, the aforementioned Sambro
The isolated segments of nucleic acids are artificially manipulated by genetic engineering techniques as described in ok ( supra ). This “recombinant nucleic acid” simply includes the addition of a part of the nucleic acid,
Also included are nucleic acids altered by substitution or deletion. A recombinant nucleic acid can include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid can be, for example, part of a vector used to transform a cell.

Alternatively, such a recombinant nucleic acid may be used for vaccination of a mammal expressing the recombinant nucleic acid and a part of a vaccinia virus-based viral vector that elicits a protective immune response in the mammal. Can be

An “RNA equivalent” for a DNA sequence is the same as the reference DNA sequence except that the nitrogenous base thymine is replaced by uracil and the sugar chain backbone consists of ribose instead of deoxyribose. Consists of a linear nucleic acid sequence.

The term “sample” is used in its broadest sense. Samples suspected of containing LBAP-encoding nucleic acids or fragments thereof, or LBAP itself, include body fluids, chromosomes isolated from cells, extracts from organelles or cell membranes, cells, in solution, or Genomic DNA, RNA or cDNA bound to a solid support, tissue, tissue prints and the like can be included.

The term “specific binding” or “specifically binds” refers to the interaction between a protein or peptide and an agonist, antibody, antagonist, small molecule or any natural or synthetic binding component. This interaction means that it depends on the presence of a particular structure of the protein (eg, an antigenic determinant or epitope recognized by the binding molecule). For example, if the antibody is specific for the epitope "A", then in the reaction involving the labeled free "A" and the antibody, the epitope "A" (ie, the free, unlabeled "A") The amount of labeled “A” that binds to the antibody is reduced by the presence of the polynucleotide containing

The term “substantially purified” refers to a nucleic acid or amino acid sequence that has been removed from its natural environment, isolated or separated from other components with which it is naturally associated. A nucleic acid or amino acid sequence whose components have been removed by at least about 60%, preferably by about 75% or more, most preferably by about 90% or more.

The term “substitution” refers to the replacement of one or more nucleotides or amino acids with another nucleotide or amino acid, respectively.

The term “substrate” refers to a membrane, filter, chip, slide, wafer, fiber,
Refers to any suitable solid or semi-solid support, including magnetic or non-magnetic beads, gels, tubes, plates, polymers, microparticles, capillaries. Substrates can take a variety of surface forms, such as walls, grooves, pins, channels, and pores to which polynucleotides and polypeptides bind.

The term “transformation” refers to a process by which exogenous DNA enters and changes a host cell. Transformation can occur under natural or artificial conditions by a variety of methods well known to those skilled in the art, and can be performed by any known method for introducing foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. Can be based on Transformation methods are selected according to the type of host cell to be transformed, including, but not limited to, viral infection, heat shock, electroporation, lipofection, and methods using microprojectile bombardment. Is included. Such "transformed" cells include stably transformed cells capable of replicating the inserted DNA autonomously or as part of the host chromosome, and limited Includes transiently transformed cells that express DNA or RNA introduced over time.

The term “genetically modified organism” includes, but is not limited to, animals and plants, wherein one or more cells of the organism have been replaced with humans using genetically modified techniques well known to those skilled in the art. Heterogeneous nucleic acids introduced by the intervention of The nucleic acid is introduced into the cell by introducing it directly or indirectly into the precursor of the cell using deliberate genetic manipulation such as microinjection or infection with a recombinant virus. The term "genetically engineered" does not include typical cross breeding or in vitro fertilization, but rather refers to introducing a recombinant DNA molecule. The genetically modified organism according to the present invention includes:
Includes bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention is
The host can be introduced by methods well known to those skilled in the art (eg, infection, transfection, transformation, or transconjugation). DNA of the present invention
Techniques for introducing E. coli into such organisms are well known, see Sambrook et al. (1989), supra.

The “variant sequence” of a specific nucleic acid sequence is defined as “BLAS” in the default parameter settings.
Using blastn with the "T2 Sequences" tool Version 2.0.9 (May-07-1999), it has been shown to have at least 40% sequence identity to a particular nucleic acid sequence at a given length. Nucleic acid sequence. Such pairs of nucleic acids may exhibit, for example, at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98% or more identity at a given length. Certain mutant sequences are, for example, "alleles" (described above).
, "Splice", "species" or "polymorphic" variant sequences. A splice variant may have significant identity to a reference molecule, but may have more or fewer polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may have additional functional domains or may lack domains present in the reference molecule. Species variant sequences are polynucleotide sequences that vary from species to species. The resulting polypeptides usually have significant amino acid identity with each other. Polymorphic variant sequences are those in which the polynucleotide sequence of a particular gene differs among individuals of a given species. Also, a polymorphic variant sequence can include a "single nucleotide polymorphism" (SNP) in which one nucleotide of a polynucleotide sequence is mutated. The presence of the SNP is, for example, a predetermined population,
It may indicate a feature of the condition or condition.

A “variant” of a particular polypeptide sequence is defined as the presence of a nucleic acid sequence using a blastp with the “BLAST 2 Sequences” tool Version 2.0.9 (May-07-1999) with default parameter settings. A nucleic acid sequence for which the identity of the particular polypeptide sequence to length has been determined to be at least 40%. A polypeptide sequence that has been determined to have at least 40% sequence identity to a particular polypeptide sequence at a given length. Such pairs of polypeptides may exhibit, for example, at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98% or more identity at a certain length. .

[0081] This invention relates to novel human leukocyte and blood-related protein (LBAP), polynucleotides encoding LBAP, and diagnosis of cell proliferative disorders, including autoimmune disorders / inflammatory diseases as well as cancer, the treatment or prevention Based on the discovery of the use of these compositions for

Table 1 shows the insight clones used to construct the full length nucleotide sequence encoding LBAP. Columns 1 and 2 show the SEQ ID NO of the polypeptide and nucleotide sequences, respectively. The third column shows the clone ID of the insight clone from which the nucleic acid encoding each LBAP was identified, and the fourth column shows the cDNA library from which those clones were isolated. Column 5 shows the insight clones and their corresponding cDNA libraries. In-site clones for which no cDNA library is shown were obtained from the pooled cDNA library. The fifth row of insight clones is used to construct a consensus nucleotide sequence for each LBAP and is useful as a fragment in hybridization techniques.

Each column of Table 2 shows various properties of each polypeptide of the present invention. The first column is SEQ ID NO, the second column is the number of amino acid residues in each polypeptide, the third column is a potential phosphorylation site, and the fourth column is a potential glycosylation site. Column 5 shows the amino acid residues having the signature sequence and motif, and column 6 shows
The homologous sequence identified by the LAST analysis is shown. The seventh column shows an analysis method, and in some cases, a searchable database that can be used by the analysis method. The analysis methods in the seventh column were used to characterize each polypeptide by sequence homology and protein motifs.

Each column of Table 3 indicates the tissue specificity, disease, disorder or condition associated with the nucleotide sequence encoding LBAP. The first column of Table 3 shows the nucleotide sequence number (SE
Q ID NO). The second column shows LBA as a percentage of all tissues expressing LBAP.
2 shows the classification of tissues expressing P. The third column shows diseases, abnormalities or symptoms associated with tissues expressing LBAP as a percentage of total tissues expressing LBAP. The fourth column shows each cD
The vector used for subcloning of the NA library is shown. Of particular note is the expression of SEQ ID NO: 10 in the primary osteogenic sarcoma cell line.

Each column in Table 4 provides a description of the tissue used to create a cDNA library from which a clone of the cDNA encoding LBAP was isolated. The first column contains the nucleotide SEQ ID NO,
The second row shows the cDNA library from which those clones were isolated, and the third row shows the cDNA library of the second row.
The origin and other explanations of the tissue related to the NA library are shown, respectively.

A fragment of the nucleotide sequence encoding LBAP shown below is, for example, SEQ ID NO:
It is useful in hybridization or amplification techniques to distinguish 6-10 from related sequences and to identify SEQ ID NO: 6-10. Its useful sequences include:
A fragment of SEQ ID NO: 6 from about nucleotide 865 to nucleotide 909;
A fragment of SEQ ID NO: 7 of the nucleotide up to position 474; a fragment of SEQ ID NO: 8 of the nucleotide from position 182 to 233; a fragment of SEQ ID NO: 9 of the nucleotide from position 408 to 452; And SEQ ID NO: about nucleotides 356 to 418.
D NO: 10 fragment is included. The polypeptides encoded by these fragments are useful, for example, as immunogenic peptides.

The present invention also includes LBAP variants. Preferred LBAP variants have at least about 80% amino acid sequence identity to the LBAP amino acid sequence, or at least about 90%, or even at least about 95%, and have a functional or structural characteristic of LBAP. Including at least one.

[0088] The invention further includes polynucleotides encoding LBAP. In certain embodiments, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 6-10 encoding LBAP. SEQ ID NO: 6-10 shown in the sequence listing
Contains an equivalent RNA sequence in which the nitrogenous base thymine is substituted with uracil and the backbone of the sugar chain is ribose instead of deoxyribose.

Further, the present invention includes a mutant sequence of the polynucleotide encoding LBAP. In particular,
The variant sequence of such a polynucleotide may be at least about 70%, or at least about 85%, or even at least about 95%, of the polynucleotide sequence encoding LBAP.
% Polynucleotide sequence identity. Certain embodiments of the present invention include SEQ ID
NO: comprises a variant sequence of a polynucleotide sequence comprising a sequence selected from the group consisting of 6-10, which is at least about 70% relative to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 6-10, Alternatively, it has at least about 85%, and even at least about 95%, polynucleotide sequence identity. All the mutant sequences of the above polynucleotides are LBAP
Can encode an amino acid sequence that includes at least one of the following functional or structural characteristics:

The degeneracy of the genetic code results in the production of a large number of LBAP-encoding polynucleotide sequences, including those with minimal similarity to known and naturally occurring gene polynucleotide sequences. Will be understood by those skilled in the art. Thus, the present invention contemplates all possible variations in the polynucleotide sequence that can be created by selecting combinations based on possible codon choices. These combinations are to be generated according to the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring LBAP, and all such variations are to be considered specifically disclosed herein.

It is preferred that the nucleotide sequence encoding LBAP and its mutant sequences are capable of hybridizing to a naturally occurring LBAP nucleotide sequence under appropriately selected stringency conditions, although it is preferred that the nucleotide sequence It is advantageous to create a nucleotide sequence encoding LBAP or a derivative thereof that has substantially different codon usage, such as including the occurrence codon. Depending on the frequency with which particular codons are utilized by the host, codons can be selected to increase the rate at which expression of the peptide occurs in eukaryotic or prokaryotic hosts. Another reason for substantially altering the nucleotide sequence encoding LBAP and its derivatives without altering the encoded amino acid sequence is that it has a longer half-life than transcripts created from naturally occurring sequences. This is to create an RNA transcript having the desired properties.

The present invention also includes the creation of DNA sequences encoding LBAP and derivatives of LBAP, or fragments thereof, exclusively by synthetic chemistry. After construction, the synthetic sequences can be inserted into any of a number of available expression vectors and cell lines using reagents well known to those of skill in the art. In addition, synthetic chemistry can be used to introduce mutations into the sequence encoding LBAP or any fragment thereof.

Also included in the invention are hybrids, under various stringent conditions, to the claimed polynucleotide sequences, and especially to SEQ ID NOs: 6-10 and fragments thereof. Includes soyable polynucleotide sequences (eg, Wahl, GM
And SL Berger (1987) Methods Enzymol. 152: 399-407; Kimmel, AR (1987) M
ethods Enzymol. 152: 507-511). Hybridization conditions include the annealing and washing conditions described in the Definitions .

[0094] DNA sequencing methods are well known and can be utilized in any of the embodiments of the present invention. The method is based on the Klenow fragment of DNA polymerase I, SEQUEN
ASE (US Biochemical, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Pharmacia Biotech, Piscataway NJ), or
Enzymes are used, such as a combination of a proofreading exonuclease and a polymerase as found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD). Prepare the array using MICROLAB 2200 liquid transfer sy
stem (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown)
It is preferred to automate by equipment such as MA) and ABI CATALYST 800 thermal cycler (Perkin-Elmer). Next, ABI 373 or 377 DNA sequencing system (Perkin-Elmer), MEGABACE 1000 DNA sequencing system (Molecul
Sequencing is performed using ar Dynamics, Sunnyvale CA) or other well-known equipment. The resulting sequence is analyzed using various algorithms well known to those of skill in the art (eg, Ausubei, FM (1997) Short Protocols in Molecular Biolog y , John Wiley & Sons, New York NY, unit 7.7; Meyers, RA (1995) Molecular Biology and Biotechnology , Wiley VCH, New York NY, pp. 856-853).

The nucleic acid sequence encoding LBAP is extended using partial nucleotide sequences in a variety of PCR-based methods well known in the art to detect upstream sequences such as promoters and regulatory elements. be able to. For example, one of the methods used, restriction site PCR, amplifies an unknown sequence from genomic DNA in a cloning vector using common primers and nested primers (eg, S
arkar, G. (1993) PCR Methods Applic. 2: 318-322). Another method that uses the inverse PCR method uses primers that extend widely and amplify the unknown sequence from the circularized template. This template is derived from a restriction fragment containing a known genomic locus and sequences around it. (Triglia, T. et al. (1988) Nucleic Acids Res. 16: 8186).
As a third method, capture PCR involves PCR amplification of a DNA fragment adjacent to a known sequence of human and yeast artificial chromosomal DNA (eg, Lag
erstrom, M. et al. (1991) PCR Methods Applic 1: 111-119). In this method, the digestion and ligation of a number of restriction enzymes can be used to insert the recombinant double-stranded sequence into a region of unknown sequence before performing PCR. Also, other methods that can be used to recover unknown sequences are well known to those of skill in the art (eg, Parker, JD
(1991) Nucleic Acids Res. 19: 3055-306). Furthermore, using PCR method, nested primers, and PROMOTERFINDER library (Clonetech, Palo Alto, CA)
Walking in genomic DNA is possible. This method eliminates the need to screen the library and is useful for finding intron / exon transitions. For all methods of PCR method was based, primer, OLIGO ^TM 4.06 Primer Analysis
Using commercially available software such as software (National Biosciences Inc., Plymouth, MN) or another suitable program, the length is 22-30 nucleotides, the GC content is about 50% or more, and about 68 ° C.-72. It can be designed to anneal to the mold at a temperature of ° C.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include large cDNAs. In addition, a random-primed library, which often contains the sequence of the 5 ′ region of a gene, is suitable when an oligod (T) library cannot obtain a full-length cDNA. Genomic libraries can also serve for extension of sequence in the 5 'non-transcribed region.

A commercially available capillary electrophoresis system can be used for size analysis or confirmation of the nucleotide sequence of the product of sequencing or PCR. In particular, capillary sequencing may use a flowable polymer for electrophoretic separation, a laser-activated fluorescent dye specific for four different nucleotides, and a CCD camera that detects the emitted wavelength. The output / light intensity is converted to an electrical signal using appropriate software (eg, GENOTYPER and SEQUENCE NAVIGATOR (Perkin Elmer)), and the entire process from sample loading to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is particularly suitable for sequencing small pieces of DNA that may be present in limited amounts in a particular sample.

In another embodiment of the present invention, a polynucleotide sequence encoding LBAP or a fragment thereof is used in a recombinant DNA molecule to express LBAP, a fragment thereof or a functional equivalent thereof in a suitable host cell. Can be oriented. Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially identical, ie, functionally equivalent, amino acid sequences are created and can be used for expression of LBAP.

The nucleotide sequence of the present invention may be modified to alter the sequence encoding LBAP by various techniques known to those of skill in the art, including, but not limited to, altering the cloning, processing and / or expression of the gene product. Operable using the method. The nucleotide sequence can be manipulated using DNA mixing by random fragmentation or PCR reconstitution of gene fragments and synthetic oligonucleotides. For example, oligonucleotide-mediated site-directed mutagenesis allows for the insertion of mutations that create new restriction sites, alter glycosylation patterns, alter codon preferences, generate splicing variants, and the like.

MOLECULARBREEDING (Maxygen Inc., Santa Clara CA;
described in US Patent Number 5,837,458; Chang, C.-C. et al. (1999) N
at. Biotechnol. 17: 793-797; Christians, FC et al. (1999) Nat. Biotechn.
ol. 17: 259-264; Crameri, A. et al. (1996) Nat. Biotechnol. 14: 315-319).
DNA shuffling techniques, such as, can be applied to alter or improve the biological or enzymatic activity of LBAP or the biological properties of LBAP, such as the ability to bind other molecules or compounds. DNA shuffling is the process of making a library of gene variants using PCR-mediated recombination of gene fragments. Next, the library is selected or screened to identify gene variants having the desired properties. These suitable mutants can then be pooled and further subjected to repeated DNA shuffling and selection / screening. Thus, genetic diversity is created by "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing irregularly mutated mutations can be recombined, screened, and re-shuffled until the desired properties are optimized. Alternatively, a given gene fragment is recombined with a homologous gene fragment of the same gene family of the same or a different species to maximize the diversity of multiple native genes in a controlled and regulatable manner. be able to.

In another embodiment of the present invention, the entire LBAP-encoding sequence, or a portion thereof, can be synthesized using chemical techniques well known to those skilled in the art (see, eg, Caruthers.
MH et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223; Horn, T. et al. (1980) Nucl.
es.Symp.Ser.225-232). Alternatively, LBAP itself or a fragment thereof can be synthesized using chemical techniques. For example, peptide synthesis can be performed using various solid phase techniques (eg, Roberge, JY et al. (1995) Science 269: 202-204).
reference). In addition, the synthesis can be automated using ABI 431A peptide synthesizer (Perkin Elmer). In addition, variant polypeptides can be produced by modifying the amino acid sequence of LBAP or any portion thereof in direct synthesis and / or binding to sequences derived from other proteins or any portion thereof. It is.

The peptide can be substantially purified by high performance liquid chromatography for separation (eg, Chiez, RM and Regnier. FZ (1990) Methods Enzymo
l. 182: 392-421). The composition of the synthesized peptide can be confirmed by amino acid analysis or sequencing (Creighton, T. (1983) Proteins. Structures avd Molecular Properties , WH Freeman, New York NY).

In order to express a biologically active LBAP, the nucleotide sequence encoding LBAP, or a derivative thereof, is required to be transferred to a suitable expression vector (ie, the transcription and translation of a coding sequence inserted into a suitable host). Vector containing the element).
These elements include regulatory sequences such as enhancers, constitutive and inducible promoters, 5 'and 3' untranslated regions in the vector and polynucleotide sequences encoding LBAP. The length and specificity of such elements can vary. Specific initiation signals can be used to effect more efficient translation of sequences encoding LBAP. Such signals include the ATG initiation codon and adjacent sequences such as, for example, the Kozak sequence. When the sequence encoding LBAP, its initiation codon, and upstream regulatory sequences are inserted into an appropriate expression vector,
Additional transcriptional and translational control signals may be unnecessary. However, if only the coding sequence or a fragment thereof is inserted, the ATG in the reading frame
Exogenous translational control signals, including the initiation codon, must be provided by the expression vector. Exogenous translational elements and initiation codons can consist of various natural and synthetic forms. Inclusion of appropriate enhancers in the particular host cell strain used can increase the efficiency of expression (see, for example, Scharf, D. et al.
994) Results Probl. Cell Differ. 20: 125-162).

To create an expression vector containing a sequence encoding LBAP and appropriate transcriptional and translational regulatory regions, methods well known to those skilled in the art are used. These methods, in vit ro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination technique (see, eg, Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Sprin
g Harbor Press, Plainview, NY, ch.4,8, and 16-17; Ausubel, FM et al. (1995) Cu rrent Protocols in Molecular Biology, John Wiley & Sons, New York, NY, c
h. 9, 13, and 16).

A variety of expression vector / host systems are available for containing and expressing sequences encoding LBAP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors, yeast transformed with yeast expression vectors, and viral expression vectors ( For example, an insect cell line infected with a baculovirus, a virus expression vector (eg, cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or a bacterial expression vector (eg, Ti or pBR322 plasmid) was used. Plant cell lines or animal cell lines are included. The present invention is not limited by the host cell utilized.

In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for the polynucleotide sequence encoding LBAP. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding LBAP can be performed using PBLUESCRIPT (Stratagene, La Jolla CA) or pSportl plasmid (Li
fe Technologies) can be performed using a multifunctional E. coli vector. Ligation of the LBAP-encoding sequence into multiple cloning sites of the vector disrupts the lacZ gene, allowing a colorimetric screening method to identify transformed bacteria containing the recombinant molecule. In addition, these vectors are useful for in vitro transcription of cloned sequences, dideoxy sequencing, rescue of single strands by helper phage, and generation of nested deletions (eg, Van Heeke. And SM Schuster (1989) J. Biol. Chem. 264: 5503-5509). For example, when a large amount of LBAP is required for the purpose of antibody production, high-level LBAP
A vector that results in the expression of For example, a vector containing a strong inducible T5 or T7 bacteriophage promoter can be used.

A yeast expression system can be used to generate LBAP. In yeast Saccharomyces cerevisiae or Pichia pastoris , a wide variety of vectors can be used, including constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH. Furthermore, such vectors direct either the secretion or intracellular retention of the expressed protein and allow for the integration of exogenous sequences into the host genome for stable growth (eg, Ausubel as described above). , supra; and Grant et al. (1987) Methods Enzymol.
153: 516-54; see Scorer, CA et al. (1994) Bio / Technology 12: 181-184).

[0108] Plant systems can also be used for expression of LBAP. Transcription of the sequence encoding LBAP can be promoted by a viral promoter, such as the CaMV 35S and 19S promoters alone or in combination with an omega leader sequence from TMV (Takamatsu, N. et al. (1987) EM).
BO J. 6: 307-311). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (For example, The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York; pp.191-196
See).

[0109] In mammalian cells, a number of viral-based expression systems are available. When an adenovirus is used as an expression vector, the sequence encoding LBAP can be ligated into an adenovirus transcription / translation complex consisting of the late promoter and tripartite leader sequence. Insertion at a non-essential E1 or E3 region of the viral genome results in an infectious virus that expresses LBAP in host cells (eg, Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. . 81: 3655-3
659). In addition, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells. Also, for high level expression of proteins, vectors based on SV40 or EBV can be used.

The use of human artificial chromosomes (HACs) also allows for the supply of larger DNA fragments than can be contained and expressed in a plasmid. For therapeutic purposes, 6 kb to 10 Mb of HAC can be constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) (eg,
Harrington, JJ et al. (1997) Nat Genet. 15: 345-355).

To ensure long-term production of recombinant mammalian proteins, stable expression of LBAP in cell lines is preferred. For example, LB using an expression vector
The sequence encoding the AP can be transformed into a cell line, the expression vector of which contains replication and / or endogenous expression elements of viral origin and a selectable marker gene on the same or a separate vector. It is. After the introduction of the vector, the cells are allowed to grow for 1-2 days in a concentrated medium before switching to a selective medium. The purpose of the selectable marker is to confer resistance to the selective mediator and to allow the growth and recovery of cells that successfully express the introduced sequence due to their presence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type.

[0112] Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, respectively, tk ^- and apr ^-. As used in the cell (e.g., Wigler, M et al (1977) Cell 11:
223-32; see Lowy, I. et al. (1980) Cell 22: 817-23). Also, resistance to antimetabolites, antibiotics or herbicides can be used as a basis for selection, for example, dhfr confers resistance to methotrexate, neo is aminoglycoside neomycin and G-418.
Als or pat are chlorsulfuron, phosphinotricin acetyltransferase
se) (eg, Wigler, M. et al. (1980) Proc. Natl. Acad. Sc
i. 77: 3567-70; see Colberre-Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14). Additional selectable genes are described in the literature, for example, trpB and hisD, which alter the cellular need for metabolites (eg, Hartman, SC and R).
.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85: 8047-8051). For example, visible markers such as anthocyanin, green fluorescent protein (GFP; Clontech), β-glucuronidase and its substrate β-glucuronide, or luciferase and its substrate luciferin can be used. These markers are widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression by a particular vector system (eg, Rhodes, CA et al. (1995) Methods Mol. Bio
l. 55: 121-131).

The presence / absence of marker gene expression also indicates the presence of the gene of interest, but may require confirmation of the presence and expression of that gene. For example, when a sequence encoding LBAP is inserted within a marker gene sequence, transformed cells containing the sequence encoding LBAP can be identified by the absence of marker gene function. Alternatively, the sequence encoding LBAP and the marker gene can be placed in tandem under the control of a single promoter. Expression of the marker gene in response to selection or induction usually indicates expression of the tandemly arranged sequence as well.

In general, nucleic acid sequences encoding LBAP may be included by various methods well known to those of skill in the art.
Host cells expressing LBAP can be identified. Such methods include, but are not limited to, DNA-DNA or DNA-RNA hybridization, PCR amplification, and membranes, solutions, or chips based on techniques for detecting and / or quantifying nucleic acid and protein sequences. Including protein bioassays or immunoassays.

Using either specific polyclonal or monoclonal antibodies
Immunological techniques for detecting and measuring LBAP expression are well known to those skilled in the art. Examples of such techniques include enzyme linked immunoassays (ELISA), radioimmunoassays (RIAs) and fluorescent cell analysis separations (FACS). Two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on LBAP
) Is preferred, but competitive binding experiments can also be used. These and other assays are disclosed by those skilled in the art (eg, Hampton, R. et al. (1990); Serological Methods, a Laboratory Manual , APS Press, St Paul, MN Section IV; Col.
igan, JE et al. (1997) Current Protocols in Immunology , Greene Pub.
tes and Wiley-lnterscience, New York, NY; and Pound, JD (1998) Immunochemical Protocols , Humana Press, Totowa NJ).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization probes or PCR probes for detecting sequences related to the polynucleotide encoding LBAP include oligolabeling (oligolabeling).
ing), nick translation, end-labeling, or PCR amplification using labeled nucleotides. Alternatively, the sequence encoding LBAP, or any fragment thereof, can be cloned into a vector for the production of an mRNA probe. Such vectors are well known to those of skill in the art and are commercially available, and can be used to
RNA probes can be synthesized in vitro by adding a suitable RNA polymerase such as, for example, T7, T3 or SP6, and labeled nucleotides. These methods are described in various commercially available kits (Amersham Pharmacia Biotech, Promega (Mad
ison WI), and US Biochemical). Suitable reporter molecules or labels used to facilitate detection include radionuclides, enzymes, fluorescent agents, chemiluminescent or dye-producing agents, substrates, cofactors, inhibitors, magnetic particles, and the like.

[0117] Host cells transformed with a nucleotide sequence encoding LBAP can be cultured under conditions suitable for the recovery and expression of the protein from cell culture.
Proteins produced by the transformed cells may be secreted or retained intracellularly depending on the sequence and / or the vector used. As will be appreciated by those skilled in the art, expression vectors containing a polynucleotide encoding LBAP can be designed to include a signal sequence that directs LBAP secretion through prokaryotic or eukaryotic cell membranes.

In addition, host cell strains are chosen for their ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation,
Glycosylation, phosphorylation, lipidation, and acylation are included. Post-translational processing that cleaves the "prepro" form of the protein can be used to identify the target, folding and / or activity of the protein. A variety of host cells (eg, CHO, HeLa, M
DCK, MEK293, and WI38) are American Type Culture Collection (ATCC, Mana
ssas, VA) and can be selected to ensure correct modification and processing of the foreign protein.

In another embodiment of the present invention, a natural, modified or recombinant nucleic acid sequence encoding LBAP is ligated to a heterologous sequence which will translate the fusion protein of any of the aforementioned host systems. I can make it. For example, a chimeric LBAP protein containing a heterologous moiety that can be identified by commercially available antibodies can facilitate screening of peptide libraries for inhibitors of LBAP activity. Also, using commercially available affinity substrates, heterologous proteins and peptide moieties can facilitate purification of the fusion protein. Such moieties include, but are not limited to, glutathione S transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c -myc, includes hemagglutinin (HA). Glutathione immobilized by GST, MBP, Trx, CBP, and 6-His,
Maltose, phenylarsine oxide, calmodulin, and metal chelating resins each allow for purification of homologous fusion proteins. FLAG, c-mc, and hemagglutinin (HA) allow for immunoaffinity purification of the fusion protein using commercially available monoclonal and polyclonal antibodies that specifically identify these epitope tags. Also, the fusion protein can be engineered such that the fusion protein contains a protein cleavage site located between the sequence encoding LBAP and the heterologous protein sequence, and the LBAP can be cleaved from the heterologous portion after purification. Methods for expression and purification of the fusion protein are described in Ausubel's literature (1995, supra , ch 10). In addition, various commercially available kits can be used to promote expression and purification of the fusion protein.

In yet another embodiment of the present invention, radiolabeled LBAP can be synthesized in vitro using a TNT rabbit reticulocyte lysate or a wheat germ extract system (Promega). These systems couple the transcription and translation of a protein-encoding sequence operably linked to a T7, T3, or SP6 promoter. Transcription takes place in the presence of a radiolabeled amino acid precursor, preferably ³⁵ S-methionine.

Not only by recombinant production, but also by direct peptide synthesis using solid phase technology,
LBAP fragments can be created (see, for example, Creighton, supra, pp. 55-60). Protein synthesis can be performed manually or automatically. Automated synthesis can be performed, for example, using an ABI 431A peptide synthesizer (Perkin Elmer). Various fragments of LBAP can be synthesized separately and then combined to create the full length molecule.

There are chemical and structural similarities, for example, in the context of sequence and motifs between therapeutic leukocytes and blood-related proteins and regions of LBAP. Furthermore, the expression of LBAP has a close relationship with cell proliferation, cancer and inflammation. Therefore, LBAP is thought to play a role in cell proliferation disorders including autoimmune disorders / inflammatory diseases and cancer. In the treatment of diseases associated with increased LBAP expression or activity, it is desirable to reduce LBAP expression or activity. In the treatment of diseases associated with decreased expression or activity of LBAP, it is desirable to increase the expression or activity of LBAP.

Accordingly, in one embodiment, LBAP or a fragment or derivative thereof can be administered to a patient for the treatment or prevention of a disease associated with reduced LBAP expression or activity. Examples of such diseases include, but are not limited to, actinic keratosis and arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MC
TD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically , Adrenal gland, bladder, bone, bone marrow, brain, breast, neck, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen Cell proliferative disorders such as cancer of the stomach, testis, thymus, and uterus; and acquired immunodeficiency syndrome (AIDS) and adrenal insufficiency, adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, and atherosclerotic artery Sclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyglandular endocrine candidal ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, Dermatomyositis, diabetes Emphysema, lymphocytes toxic temporary lymphocytes thrombocytopenia, erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout,
Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenia, ulcerative colitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation And autoimmune / inflammatory diseases such as viral and bacterial infections, fungal infections, parasite infections, protozoal infections, helminth infections, trauma, and the like.

In another embodiment, a vector capable of expressing LBAP or a fragment or derivative thereof for the treatment or prevention of a disease associated with reduced LBAP expression or activity, including, but not limited to, those described above. It may be administered to a patient.

In another embodiment, purified LBAP with a suitable pharmaceutical carrier is used to treat or prevent a disease associated with reduced LBAP expression or activity, including but not limited to those described above. May be administered to a patient.

In yet another embodiment, an agonist that modulates LBAP activity is administered to a patient for the treatment or prevention of a disease associated with reduced LBAP expression or activity, including but not limited to those described above. May be.

In yet another embodiment, an LBAP antagonist may be administered to a patient for the treatment or prevention of a disease associated with increased LBAP expression or activity. Examples of such diseases include, but are not limited to, the autoimmune disorders / inflammatory diseases described above and cell proliferation disorders, including cancer. In one embodiment, an antibody that specifically binds to LBAP can be used directly as an antagonist or indirectly as a delivery mechanism or targeting to bring the agent to cells or tissues that express LBAP.

In another embodiment, the complementary sequence of a polynucleotide encoding LBAP can be expressed for the treatment or prevention of a disease associated with increased LBAP expression or activity, including but not limited to those described above. The appropriate vector may be administered to the patient.

In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences or vectors of the invention may be administered in combination with other suitable therapeutic agents. One of skill in the art would be able to select appropriate agents for use in combination therapy based on conventional pharmaceutical principles. The combination of therapeutic agents can function synergistically to effect treatment or prevention of the various reactions described above. By using this method, the therapeutic effect can be increased with a smaller dose of each drug, and thus the possibility of side effects can be reduced. Antagonists of LBAP can be produced using methods well known to those skilled in the art. In particular, libraries of drugs can be screened to generate antibodies using purified LBAP or to identify those that specifically bind to LBAP. Antibodies to LBAP can be produced using methods well known to those skilled in the art. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries. Neutralizing antibodies (ie, those that inhibit dimer formation) are particularly suitable for therapeutic use.

To raise antibodies, goats, rabbits, rats, mice, or mice can be injected by injecting LBAP or any fragment thereof having immunological properties or oligopeptides thereof to produce antibodies.
Various hosts including humans can be immunized. Rats and mice are preferred as hosts for downstream applications involving monoclonal antibody production.
Depending on the species of the host, various adjuvants can be used to enhance the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH And dinitrophenol. Among adjuvants used in humans, BCG ( bacilli Calmette-Guerin) and Coryn ebacterium parvum are particularly preferred.

The oligopeptide, peptide or fragment thereof used to elicit antibodies for LBAP has an amino acid sequence consisting of at least 5 amino acids, and generally at least 10 amino acids. It is also preferred that these oligopeptides, peptides or fragments are identical to part of the amino acid sequence of the natural protein and contain the entire amino acid sequence of the small natural molecule. The short extension part of LBAP amino acid is KLH
Can be fused to an extension of another protein, such as, to produce chimeric molecule antibodies.

[0132] Monoclonal antibodies to LBAP can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. Such techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology (eg, Kohler, G. et al. (1975) Nature 256: 495-497).
Kozbor, D. et al. (1985) Immunol. Methods 81: 31-42; Cote, RJ et al. (1983) Pr.
oc. Natl. Acad. Sci. 80: 2026-2030; Cole, SP et al. (1984) Mol. Cell Biol. 6
2: 109-120).

In addition, techniques developed for the production of “chimeric antibodies”, such as the technique of splicing a mouse antibody gene to a human antibody gene, in order to obtain molecules with appropriate antigen specificity and biological activity (For example, Morrison, SL et al. (
Natl.Acad.Sci. 81: 6851-6855; Neuberger, MS et al. (1984) Nature.
312: 604-608; Takeda, S. et al. (1985) Nature 314: 452-454). Alternatively, techniques for the production of single-chain antibodies can be applied using methods well known to those skilled in the art to produce single-chain antibodies specific for LBAP. Antibodies with related specificities but different idiotypic compositions can be produced by chain shuffling from random combinations of immunoglobulin libraries (eg, Burton DR).
(1991) Proc. Natl. Acad. Sci. 88: 10134-10137).

Antibodies can be produced by screening an immunoglobulin library or panel of highly specific binding reagents, or by inducing production in a lymphocyte population in vivo (eg, Orlandi, R. et al. (1989), Proc. Natl
Acad. Sci. 86: 3833-3837; see Winter, G. et al. (1991) Nature 349: 293-299).

[0135] Antibody fragments which contain specific binding sites for LBAP can also be generated. For example, such fragments include, but are not limited to, F (ab ') ₂ fragments that can be generated by pepsin digestion of antibody molecules, and F (ab') ₂ fragments that are generated by reducing disulfide bridges. Fab fragments that can be used. Alternatively, a Fab expression library can be constructed so that monoclonal Fab fragments with the desired specificity can be identified quickly and easily (see, for example, Huse, WD et al. (1989) Science 246: 1275-1281).

[0136] A variety of immunoassays can be used for screening to identify antibodies with the desired specificity. Numerous protocols for competitive binding assays or immunoradiometric assays using either monoclonal or polyclonal antibodies with established specificity are well known in the art. Such immunoassays generally involve the measurement of the formation of a complex between LBAP and its specific antibody. Use monoclonal antibodies reactive against two non-interfering LBAP epitopes2
Two sites monoclonal base-based immunoassay
d immunoassay) is usually used, but competitive binding assays can also be used (Po
und, supra ).

Using various methods such as Scatchard analysis with radioimmunoassay technology, LBA
Evaluate the affinity of the P antibody. Affinity is expressed as the binding constant, Ka, defined as the molar concentration of the OP antibody complex at equilibrium divided by the molar concentrations of free antibody and free antigen. The Ka measured for a polyclonal antibody reagent with heterogeneous affinities for a number of LBAP epitopes represents the average affinity or avidity of the LBAP antibody. The Ka measured for a monoclonal antibody reagent monospecific for a particular LBAP epitope represents a true measure of affinity. High affinity antibody reagents with Ka values of 10 ⁹ to 10 ¹² L / mol are preferably used in immunoassays where the LBAP antibody complex must withstand rigorous manipulations. Low-affinity antibody reagents with a Ka value of 10 ⁶ to 10 ⁷ L / mol are required for immunopurification (immunopur
) and similar treatments. (Catty, D. (1988) Antibod ies, Volume I:. A Practical Approach IRL Press, Washington, DC; Liddell,
JE and Cryer, A. (1991) A Practical Guide to Monocional Antibodies , J
ohn Wiley & Sons, New York NY).

The titer and binding activity of the polyclonal antibody reagents are further evaluated to determine the quality and suitability of such reagents for certain downstream applications. For example, a polyclonal antibody reagent containing at least 1-2 mg / ml of specific antibody, preferably 5-10 mg / ml of specific antibody, is commonly used in methods requiring precipitation of the LBAP antibody complex. . Treatments for antibody specificity, titer, and avidity in various applications, as well as guidelines for antibody quality and usage are generally available (
For example, Catty, supra, and Coligan et al., Supra).

In another embodiment of the invention, a polynucleotide encoding LBAP, or any fragment or complement thereof, can be used for therapeutic purposes. In one aspect, the mRNA
In situations in which it is desirable to inhibit the transcription of LBAP, a complementary sequence to a polynucleotide encoding LBAP can be used. In particular, cells can be transformed with sequences complementary to a polynucleotide encoding LBAP. Therefore, LBAP
Complementary molecules or fragments can be used to modulate the activity of, or to regulate gene function. At present such techniques are well known and sense or antisense oligonucleotides, or larger fragments, can be designed from various locations according to the coding and regulatory regions of the sequence encoding LBAP.

Expression vectors derived from retroviruses, adenoviruses, herpes or vaccinia viruses, or from various bacterial plasmids, are used for delivery of nucleotide sequences to target organs, tissues, or cell populations. obtain. Vectors for expressing a nucleic acid sequence complementary to a polynucleotide encoding LBAP can be produced using methods well known to those of skill in the art (see, for example, Sambrook, supra , and
Ausubel, 1995, supra ).

A gene encoding LBAP can be created by transforming a cell or tissue with an expression vector that expresses a polynucleotide or fragment thereof encoding LBAP at high levels. Such constructs can be used to introduce non-translatable sense or antisense sequences into cells. Even in the absence of integration into DNA, such vectors can continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression can last for a month or more with non-replicating vectors, and even longer if appropriate replication elements are part of the vector system.

As described above, altering gene expression by designing a sequence complementary to the control, 5 ', or regulatory regions of the gene encoding LBAP, ie, an antisense molecule (DNA, RNA or PNA), can be performed. it can. Transcription start point (eg, +1 from start site)
Oligonucleotides derived from positions 0 to -10) are preferred. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Polymerase,
Triple helix pairing is useful because it inhibits the ability of the double helix to unwind sufficiently for binding of transcription factors or regulatory molecules (eg, Gee, JE et al. (1994) In:
Huber, BE and BI Carr, Molecular and Immunologic Approaches , Futura
Publishing Co., Mt. Kisco, NY, pp. 163-177). Complementary sequences, ie, antisense molecules, can be designed to inhibit mRNA transcription by blocking transcript binding to ribosomes.

A ribozyme, an enzymatic RNA molecule, can be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves specific hybridization of the sequence of the ribozyme molecule to complementary target RNA, followed by nucleotide strand breaks. For example, engineered hammerhead motif ribozyme molecules can specifically and effectively catalyze nucleotide strand breaks in sequences encoding LBAP.

A specific ribozyme cleavage site within any potential RNA target is first identified by scanning the target molecule for a ribozyme cleavage site that includes the following sequences GUA, GUU and GUC. Once identified, a short RNA sequence between 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site is a secondary structural feature that renders the oligonucleotide inoperable. Can be evaluated. The suitability of the candidate target can also be assessed using a ribonuclease protection assay by testing for accessibility to hybridization with the complementary oligonucleotide.

The complementary ribonucleic acid molecules and ribozymes of the present invention can be created by methods well known in the art for synthesizing nucleic acid molecules. These include techniques for the chemical synthesis of oligonucleotides, such as solid phase phosphoramidite chemical synthesis. Alternatively, the RNA molecule can be a DNA sequence encoding LBAP in vitro and in vivo.
Can be created by the transfer of Such a DNA sequence can be incorporated into a variety of vectors with an appropriate RNA polymerase promoter, such as T7 or SP6. Alternatively, these cDNA constructs that synthesize constitutively or inducibly complementary RNA can be introduced into cell lines, cells or tissues.

[0146] RNA molecules can be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'and / or 3' end of the molecule, phosphorothioate or 2'O instead of phosphodiesterase linkages in the backbone of the molecule. -Including using methyl. This concept is unique in PNA production, as well as acetyl-, methyl-, thio-, and similar modified forms of adenine, cytidine, guanine, thymine, and uridine that are not readily recognized by endogenous endonucleases. It can be extended to all these molecules by including less commonly used bases such as, inosine, queosin, and wibutosin.

A number of methods are available for introducing vectors into cells or tissues, and are also suitable for use in vivo , in vitro , and ex vivo . For ex vivo therapy, the vector can be introduced into stem cells taken from a patient and propagated as a clone for autologous transplantation and return to the same patient. Delivery by transfection or delivery by liposome injection or polycationic amino polymer can be performed using methods well known in the art (eg, Goldman, CK et al. (1997) Nature Biotechnology 15: 462-). 466)
.

Any of the above treatments can be applied to any subject in need of treatment, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits and monkeys.

In a further embodiment of the invention, the pharmaceutical ingredient is administered with a pharmaceutically acceptable carrier, for any of the therapeutic effects described above. Such pharmaceutical ingredients may consist of LBAP, antibodies of LBAP, mimetics of LBAP, agonists, antagonists or inhibitors. The component is administered alone or in combination with one or more other agents, such as a stabilizing formulation, to any sterile, biocompatible pharmaceutical carrier, which includes, but is not limited to, saline , Buffered saline, glucose or water. Such compositions may be administered to the patient alone or in combination with other drugs, drugs or hormones.

The route of administration of the pharmaceutical composition used in the present invention is not limited to the following, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal,
Intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration may be included.

In addition to the active ingredient, these pharmaceutical ingredients include suitable pharmaceutically acceptable carriers, including excipients and adjuvants that facilitate the processing of the active ingredient into pharmaceutically usable formulations. sell. Further details on techniques for formulation or administration can be found in the latest edition of Remingtons Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).

Pharmaceutical compositions for oral administration are formulated using pharmaceutically acceptable carriers well known in the art and in dosages suitable for oral administration. With such a carrier, the pharmaceutical composition can be prepared into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like for ingestion by a patient.

Pharmaceutical preparations for oral administration are prepared by mixing the active ingredient with solid excipients and treating the resulting granule mixture (after pulverization, if desired) to prepare tablets or dragee cores.
gee core). If necessary, suitable additives can be added. Suitable excipients include sugar or protein excipients such as lactose, sucrose, mannitol or sugars including sorbitol, corn,
Starches from wheat, rice, potatoes and the like, celluloses such as methylcellulose, hydroxypropylmethylcellulose or sodium carboxymethylcellulose, gums such as gum arabic or tragacanth, and proteins such as gelatin or collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof such as sodium alginate.

Dragee cores are used with suitable shells, such as concentrated sugar solutions, and include gum arabic, talc, polyvinylpyrrolidone, carbopol gel.
l), polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures and the like. Dyestuffs or pigments may be added to the tablets or dragees for tablet identification or to characterize the amount of active ingredient (ie, dosage).

Preparations that can be administered orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a tablet, such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient in admixture with excipients or binders such as lactose or starch, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid or liquid polyethylene glycol, with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active ingredients may be prepared as appropriate oily injection suspensions.
Suitable lipophilic solvents or excipients include fatty oils such as sesame oil, and synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Also, non-lipid polycationic amino polymers are used for delivery. If desired, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are well-known in the art.

The pharmaceutical composition of the present invention can be prepared by a known method, for example, a usual mixing treatment, dissolution treatment, granulation treatment, sugar coating formation treatment, wet grinding treatment (levigating), emulsification treatment, encapsulation treatment,
It is manufactured by entrapping or freeze-drying. The pharmaceutical composition may be provided as salts and may be formed with a number of acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or protonic solvents than the corresponding free base forms. In another case, a suitable formulation would be 1 mM to 50 mM histidine, 0.1% to 2% sucrose, 2% to 7% mannitol, buffered before use, in the range of pH 4.5 to 5.5. A freeze-dried powder containing any or all of them may be used.

After the pharmaceutical composition has been prepared, it can be placed in a suitable container and labeled for treatment of an indicated condition. In the case of LBAP administration, such labels include
The dosage, administration frequency, and administration method are displayed.

Pharmaceutical compositions suitable for use in the present invention include those that contain the active ingredient in an effective amount to achieve the desired purpose. Determination of an effective amount can be made well within the capabilities of those skilled in the art.

For any compound, the therapeutically effective amount can be estimated initially either in animal models, such as, for example, mice, rabbits, dogs, pigs, or in cell culture assays of neoplastic cells. In addition, animal models can be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine dosages and routes for administration in humans.

A therapeutically effective amount is, for example, LBAP or a fragment thereof that ameliorates a symptom or condition,
It refers to the amount of an active ingredient of an antibody of LBAP, an agonist of LBAP, an antagonist of LBAP, or an inhibitor of LBAP. Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED ₅₀ (therapeutically effective dose in 50% of the population) or LD ₅₀ (lethal dose for 50% of the population Amount) can be determined by calculating the statistic. The dose ratio of toxic to therapeutic effects is the therapeutic index and the LD ₅₀ / E
It can be expressed as a _D50 ratio. Preferably, the pharmaceutical composition exhibits a large therapeutic index. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for human use. The dosage of such components is less toxic or no toxicity is preferably in the range of concentrations circulating containing ED _50. Depending on the dosage form used, the sensitivity of the patient and the route of administration,
Dosage will vary within this range.

The exact dosage will be determined by the practitioner, in light of factors related to the patient that requires treatment.
Dosage and administration are adjusted to provide sufficient levels of the active ingredient, ie, to maintain the desired effect. Factors to consider include disease severity, general patient health, age, weight, gender, diet, time and frequency of administration, concomitant medications,
Includes response sensitivity, and response to treatment. Long-acting pharmaceutical compositions may be given every 3 to 4 days, every week, or 2 or 3 days depending on half-life and clearance rate of the particular formulation.
It may be administered once a week.

Normal dosage amounts range from 0.1 to 100,000 μg, and vary with the route of administration, up to a total dose of about 1 g. Guidance on specific dosages or methods of delivery is provided in the literature and is generally available to physicians in the art. One skilled in the art can employ different formulations for nucleotides than for proteins and inhibitors. Similarly, delivery of a polynucleotide or polypeptide can be specific to a particular cell, condition, location, etc.

Diagnosis In another embodiment, an antibody that specifically binds to LBAP may be used to diagnose a disease characterized by LBAP expression or to treat a patient being treated with LBAP or an agonist, antagonist or inhibitor of LBAP. It can be used in assays for monitoring. Antibodies useful for diagnostic purposes can be made in a manner similar to that for therapeutics described above. LBAP diagnostic assays include human body fluids,
Methods include the use of antibodies and labels to detect LBAP in cell or tissue extracts. Antibodies can be used with or without modification, and can be labeled by binding, either covalently or non-covalently, to a reporter molecule. A variety of reporter molecules known to those of skill in the art may be used, some of which are described above.

A variety of protocols for measuring LBAP, including ELISA, RIA and FACS, are well known in the art and provide the basis for diagnosing changes in LBAP expression and levels of abnormalities . The normal, or standard, value of LBAP expression is the binding of LBAP antibodies to body fluids or cell extracts obtained from normal mammalian (eg, human) patients under conditions suitable for complex formation. Can be established. The standard amount of complex formation can be quantified using various methods such as photometric means. The amount of LBAP expressed in control and diseased samples from the patient's biopsy tissue is compared to a standard value. Establish parameters for disease diagnosis by deviation between standard values and patient values.

In another embodiment of the present invention, a polynucleotide encoding LBAP can be used for diagnostic purposes. Polynucleotides used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. This polynucleotide is used to detect or quantify gene expression in biopsy tissue where LBAP expression may be associated with disease. Diagnostic assays can be used to identify the presence, absence, or overexpression of LBAP and to monitor regulation of LBAP levels during therapeutic treatment.

In one embodiment, identifying nucleic acid sequences encoding LBAP using hybridization with a PCR probe capable of detecting a polynucleotide sequence, including genomic sequences, encoding LBAP or a closely related molecule may be used. it can. The specificity of the probe, ie, the region in which the probe is highly specific (eg, the 5 'regulatory region)
Or the specificity of the hybridization or amplification (maximum, high, medium, or low) depending on whether it is derived from a less specific region (eg, a conserved motif). Depending on the sex, the probe
To determine only the naturally occurring sequence that encodes, or alleles and related sequences.

Probes can also be used to detect related sequences, and should preferably have at least 50% sequence identity to any sequence encoding LBAP. The hybridization probe of the present invention may be derived from DNA or RNA, or derived from the sequence of SEQ ID NO: 6-10, or derived from the sequence of the genome including introns, enhancers, and promoters of the LBAP gene. .

Means for generating specific hybridization probes for DNA encoding LBAP include cloning a polynucleotide sequence encoding LBAP or an LBAP derivative into a vector to create an mRNA probe. is there. Such vectors are well known to those skilled in the art and are commercially available, and can be used to synthesize RNA probes in vitro by adding an appropriate RNA polymerase or an appropriate labeled nucleotide. Hybridization probes can be labeled by various reporter groups, for example, the label can be from a radionuclide such as ³² P or ³⁵ S, or an enzyme such as alkaline phosphatase that binds to the probe via an avidin / biotin binding system. And the like.

A polynucleotide sequence encoding LBAP can be used for diagnosis of a disease associated with LBAP expression. Examples of such diseases include, but are not limited to, actinic keratosis and arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), bone marrow Fibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically, adrenal gland, Bladder, bone, bone marrow, brain, breast,
Cell proliferation disorders such as cervical, gallbladder, ganglion, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus, uterine cancer; And acquired immunodeficiency syndrome (AIDS) and adrenal insufficiency, adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroid Inflammation, autoimmune polyglandular endocrine candidal ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis,
Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, lymphocotoxin transient lymphopenia, erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves Disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, lighter Syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenia, ulcerative colitis, Werner syndrome, cancer complications,
Includes hemodialysis, extracorporeal circulation, and autoimmune / inflammatory disorders such as viral and bacterial infections, fungal infections, parasitic infections, protozoan infections, helminth infections, trauma, and the like. The LBAP-encoding polynucleotide sequence can be analyzed by Southern blot or northern analysis using patient biopsy tissue or body fluid, dot blot or other membrane-based techniques, PCR techniques, dipstick assays (dipstick assays). ), Pin and multiformat ELISA-like assays and microarrays can be used to detect changes in LBAP expression. Such qualitative or quantitative test methods are well known to those skilled in the art.

In certain embodiments, nucleotide sequences encoding LBAP may be useful, particularly in assays that detect the presence of a related disorder as described above. The nucleotide sequence encoding LBAP can be labeled by standard techniques and added to a body fluid or tissue sample from a patient under conditions suitable for forming hybridization complexes.
After an appropriate incubation period, the sample is washed and the signal is quantified and compared to a standard value. If the amount of signal in the patient sample is significantly altered relative to the control sample, an altered level of the nucleotide sequence encoding LBAP in the sample is indicative of the presence of the associated disease. Such assays can also be used to evaluate the effectiveness of a particular therapeutic treatment in animal studies, clinical trials, or monitoring the treatment of an individual patient.

To provide a basis for the diagnosis of diseases associated with LBAP expression, a normal, ie, standard, expression profile is established. This is accomplished by combining a body fluid or cell extract from a normal animal or human patient with a sequence encoding LBAP or a fragment thereof under conditions suitable for hybridization or amplification. Standard hybridization is based on values obtained from normal patients,
It can be quantified by comparing with values obtained from experiments using known amounts of substantially purified polynucleotide. The standard values thus obtained from a normal sample can be compared to values obtained from a sample of a patient with signs of disease. Deviation from standard values is used to prove the presence of the disease.

[0174] Once the presence of the disease is confirmed and the treatment protocol is initiated, the hybridization assay is repeated periodically to determine whether the level of expression in the patient has begun to approach that observed in normal patients. Can be evaluated. The results from successive assays can be used to show the efficacy of treatment over days to months.

With respect to cancer, the presence of abnormal amounts of transcripts (insufficient or overexpressed) in biopsy tissue from an individual indicates a predisposition to the development of the disease, ie, the actual clinical symptoms are It can be a means to detect disease before it appears. This type of more definitive diagnosis allows health professionals to take preventive measures and provide more aggressive treatment earlier, thus preventing the development and further progression of cancer .

[0176] Additional diagnostic uses for oligonucleotides designed from sequences encoding LBAP include the use of PCR. These oligomers may be chemically synthesized, made with enzymes, or made in vitro . The oligomer preferably comprises a fragment of a polynucleotide encoding LBAP, or a fragment of a polynucleotide complementary to a polynucleotide encoding LBAP, under optimized conditions to identify a particular gene or condition. Used. Oligomers can also be used under mild stringency conditions for the detection and / or quantification of closely related DNA or RNA sequences.

Methods used to quantify LBAP expression also include the use of radiolabeled or biotinylated nucleotides, the use of co-amplification of control nucleic acids, and the use of experimental results interpolated with a standard calibration curve. (For example, Melby, PC et al. (
(1993) J. Immunol. Methods, 159: 235-244; Duplaa, C. et al. (1993) Anal. Bioche.
m. 229-236). The rate of quantification of large numbers of samples is determined by ELISA where the oligomer of interest is present in various dilutions and is quickly quantified by spectrophotometric or colorimetric response.
It can be accelerated by performing a formal assay.

In another embodiment, oligonucleotides or longer fragments from any of the polynucleotide sequences disclosed herein can be used as targets in a microarray. Microarrays can be used to simultaneously monitor the expression levels of many genes and identify mutated sequences, mutations, and polymorphisms in genes. This information is useful in determining gene function, understanding the genetic basis of disease, diagnosing disease, and developing therapeutics and monitoring their activity.

Microarrays may be prepared and used to analyze by methods well known to those of skill in the art (eg, Brennan, TM et al. (1995) US Patent NO. 5,474,796; Schena, M., et al.
(1996) Proc. Natl. Acad. Sci. 93: 10614-10619; Baldeschweiler et al. (1995) P
CT application WO95 / 251116; Shalom D. et al. (1995) PCT application WO95 / 355
05: Heller. RA et al. (1997) Proc. Natl. Acad. Sci. 94: 2150-2155; and Hel.
ler. MJ et al. (1997) US Patent No. 5,605,662).

In another embodiment of the present invention, nucleic acid sequences encoding LBAP can be used to create hybridization probes useful for mapping naturally occurring genomic sequences. This sequence can be mapped to a particular chromosome, a particular portion of the chromosome, or an artificial chromosome construct, including, for example, human artificial chromosomes (HACs), yeast artificial chromosomes (YACs) , Bacterial artificial chromosomes (
BACs), bacterial P1 constructs or single-stranded chromosomal cDNA libraries (eg, Harr
ington et al. (1997) Nat Genet. 15: 345-355; Price, CM (1993) Blood Rev.
7: I27-134; and Trask. BJ (1991) Trends Genet. 7: 149-154).

Fluorescent in situ hybridization (FISH) may be relevant to other physical chromosome mapping techniques and genetic map data (eg, Heinz-Ulrich et al. (1995) in M.
eyers, supra , pp. 965-968). Examples of genetic map data can be found in various scientific journals or in the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. The location of the LBAP-encoding sequence on the physical chromosomal map and its association with a particular disease, or with a predisposition to a particular disease, helps to define the region of DNA involved in the disease. The nucleotide sequence of the present invention can be used to detect differences in gene sequences between normal individuals, carriers, and affected individuals.

To expand the genetic map, physical mapping techniques such as in situ hybridization of chromosomal constructs and linkage analysis using established chromosomal markers can be used. In many cases, even if the number or arm of a particular human chromosome is unknown, the placement of the gene on the chromosome of another mammal, such as a mouse, will reveal relevant markers. By physical mapping, new sequences can be assigned to chromosomal arms, or parts thereof. This can provide valuable information to researchers searching for disease genes using positional cloning or other gene discovery techniques. Once a disease or syndrome has been identified in a particular genomic region (eg, 11q22-
Once incompletely positioned by the genetic linkage to telangiectasia to 23), any sequence mapped to that region may represent a relevant or regulatory gene for further study ( For example, Gatti, RA et al. (1988) Nat
ure 336: 577-580). In addition, using the nucleotide sequence of the present invention, it is also possible to detect a difference between the position of a chromosome of a normal person and the position of a chromosome caused by translocation, inversion or the like of a carrier or an affected individual.

In another embodiment of the present invention, LBAP or a catalytic or immunogenic fragment thereof,
Alternatively, those oligopeptides can be used for screening libraries of compounds in various drug screening techniques. The fragments used in such a screen can be free in solution, attached to a solid support, attached to a cell surface, or located intracellularly. The formation of a binding complex between LBAP and the agent to be tested can be measured.

Another drug screening technique provides high-throughput screening for compounds with appropriate binding affinity for the protein of interest (eg,
(See Geysen et al. (1984) PCT application WO 84/03564). In this method, many different small test compounds are synthesized on a solid substrate. The test compound is reacted with LBAP or a fragment thereof and washed. Next, bound LBAP is detected by methods well known in the art. Also, purified LBAP can be coated directly on a plate for use in the aforementioned drug screening techniques. Alternatively, the peptide can be captured and immobilized on a solid support using a non-neutralizing antibody.

In another example, a competitive drug screening assay can be used in which neutralizing antibodies capable of binding LBAP specifically compete with the test compound for LBAP binding. In this way, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with LBAP.

In yet another embodiment, the new technology is based on the properties of currently known nucleotide sequences, including, but not limited to, the triplet genetic code and specific base pairing interactions. If available, the nucleotide sequence encoding LBAP can be used for molecular biology techniques that have not yet been developed.

[0187] Those skilled in the art will be able to utilize the present invention with the foregoing description without further explanation. Therefore, the specific preferred embodiments described below are merely illustrative and do not limit the invention.

All patents, patent applications and publications mentioned herein, particularly US patent application Ser.
No. 22,080 is hereby incorporated by reference.

[0189]

【Example】

1 Preparation of cDNA library RNA was purchased from Clontech or isolated from the tissues shown in Table 4. Some tissues are homogenized and dissolved in a guanidinium isothiocyanate solution, while other tissues are homogenized and dissolved in phenol, or TRIZOL (Life Technologies), guanidinium isothiocyanate and phenol alone. Dissolved in a mixture of suitable denaturants, such as a phase solution. The resulting lysate was centrifuged on a CsCl cushion or extracted with chloroform. RNA was precipitated from the lysate with either isopropanol or sodium acetate and ethanol, or another conventional method.

Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity.
In some cases, RNA was treated with DNase. Oligo for most libraries
d (T) -bound paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatswor
th CA) or OLIGOTEX mRNA purification kit (QIAGEN) to isolate poly (A +) RNA. Alternatively, RNA was isolated directly from tissue lysates using another RNA isolation kit such as, for example, the POLY (A) PURE mRNA purification kit (Ambion, Austin TX).

In some cases, RNA was provided to Stratagene to create a corresponding cDNA library. If not, cDNA was synthesized using the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies) by a method known to those skilled in the art or by a similar method (eg, Ausubel, 1997, supra , units 5.1-6.6). Reverse transcription was initiated using oligo d (T) or random primers. After binding of the synthetic oligonucleotide adapter to the double-stranded cDNA, the cDNA was digested with the appropriate restriction enzyme (s). Most libraries use SEPHACRYL S1000, SEPHAROSE CL2B, or SE
The size of cDNA (300-1000 bp) was selected by PHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or agarose gel electrophoresis for separation. Suitable plasmids (eg, PBLUESCRIPT plasmid (Stratagene), pSP
ORT1 plasmid (Life Technologies), pcDNA2.1 plasmid (Invitrogen, Carlsb
ad CA) or pINCY plasmid (Incyte Pharmaceuticals, Palo Alto CA) and the like. Recombinant plasmid, St
Competent E. coli cells containing XL1-Blue, XL1-BlueMRF, or SOLR from ratagene, or DH5α, DH10B, or ElectroMAX DH10B from Life Technologies were transformed.

2 Isolation of cDNA Clones Plasmids were recovered from host cells by excision in vivo using the UNIZAP vector system (Stratagene) or cell lysis. Magic or WIZARD Mini
preps DNA Purification System (Promega), AGTC Miniprep Purification Kit (Edge Biosyste
ms, Gaithersburg MD) and QIAGEN's QIAWELL 8 Plasmid, QIAWELL 8 Plus P
Plasmids were purified using at least one of lasmid, QIAWELL 8 Ultra Plasmid purification system or REAL Prep 96 plasmid kit (QIAGEN). After precipitation, they were resuspended in 0.1 ml of distilled water and stored at 4 ° C. with or without lyophilization.

Alternatively, plasmid DNA was amplified from host cell lysates by direct binding PCR in a high-throughput format (Rao, VB (1994) Anal. Blochem. 216: 1-1).
Four). The lysis and thermal cycling process of the host cells was performed in a single reaction mixture. Samples are processed and stored in 384-well plates and the concentration of amplified plasmid DNA is measured using PICOGREEN dye (Molecular Probes, Inc., Eugene, OR) and a Fluoroskan II fluorescence scanner (Labsystems Oy, Helsinki, Finland). It was measured quantitatively.

3 Sequencing and Analysis The cDNA sequencing reaction was performed using standard methods or ABI CATALYST 800 (Perki
n-Elmer) High-throughput equipment such as thermal cycler or PTC-200 thermal cycler (MJ Research) and HYDRA microdispenser (Robbins Scientific)
Alternatively, the treatment was performed using MICROLAB 2200 (Hamilton) liquid transfer system. The cDNA sequencing reaction was performed using reagents provided by Amersham Pharmacia Biotech or ABI PRISM BIGDYE Terminator cycle sequencing ready.
It was prepared using the reagents supplied in an ABI sequencing kit such as a reaction kit (Perkin-Elmer). Electrophoretic fractionation of cDNA sequencing reactions and detection of labeled polynucleotides were performed using the MEGABACE 1000 DNA Sequencing System (Molecular Dynamics); ABI PRISM 373 or 377 Sequencing System (Perkin-Elmer) and standard ABI protocols. Combination with base pairing software; or other sequence analysis systems well known to those skilled in the art. cDNA
Reading frames in the sequence were identified using standard methods (Ausubel, 1997, supra , uni
t 7.7 review). Some of the cDNA sequences were selected and extended as described in 5 of this example.

Using a combination of software programs that utilize well-known algorithms,
Polynucleotide sequences obtained from NA sequencing were constructed and analyzed. Table 5 shows a summary of the tools, programs, and algorithms used, along with appropriate descriptions, references, and threshold parameters. The first column of Table 5 is the tools, programs, and algorithms used, the second column is a brief description of them, and the third column is a citation (all of which are incorporated herein by reference. ), And the fourth column contains the appropriate score, probability value,
And other parameters (the higher the score, the higher the homology between the two sequences).
The sequence was obtained from MACDNASIS PRO software (Hitachi Software Engineering, South S
an Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and polypeptide sequence alignments are performed using the MEGALIGN multi-sequence alignment program (DNASTAR), which calculates the percent identity between aligned sequences.
) Was created using the default parameters specified by the clustal algorithm incorporated in.

Validation of polynucleotide sequences can be accomplished by removing vectors, linkers and polyA sequences and masking ambiguous base pairs using programs and algorithms based on BLAST, dynamic programming and dinucleotide nearest neighbor analysis. It was performed by doing. The sequences were then sequenced using GenBank primate, rodent, mammalian, vertebrate and eukaryotic databases using programs based on BLAST, FASTA and BLIMPS, and BLOCKS, PRINN.
Selected sequences from public databases such as TS, DOMO, PRODOM and PFAM were queried to obtain annotations. Sequences were assembled into full-length polynucleotides using programs based on Phred, Phrap and Consed and screened in reading frame using programs based on GeneMark, BLAST and FASTA. The full-length polynucleotide sequence is translated to derive the corresponding full-length amino acid sequence, which is then translated into the GenBank database (described above), SwissProt, BLOCKS, PRIN
Analyzed by querying protein family databases such as TS, DOMO, PRODOM, Prosite and PFAM based on Hidden Markov Model (HMM). HMM
Is a probabilistic approach to analyzing the common primary structure of gene families (see, eg, Eddy, SR (1996) Cur. Opin. Str. Biol. 6: 361-365).

The programs described above for the construction and analysis of full-length polynucleotide and amino acid sequences were also used to identify fragments of the polynucleotide sequence from SEQ ID NO: 6-10. Fragments of about 20 to about 4000 nucleotides useful for hybridization and amplification have been described above in the " Invention " section.

4 Northern Analysis Northern analysis is an experimental technique used to detect the presence of gene transcripts, where a membrane bound to RNA from a particular cell type or tissue is labeled and labeled. Involved in hybridization with nucleotide sequences (eg,
Sambrook et al., Supra, ch.7; supra and Ausubel, FM et al., See ch.4 and 16).

Using a similar computer technique using BLAST, GenBank or LIFESEQ (I
Search for identical or related molecules in nucleotide databases such as ncyte Pharmaceuticals). This analysis is faster than many membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is classified as an exact match or similar. The search criteria are based on the product score defined below.
). (% Sequence match x% max BLAST score) / 100 The product score takes into account both the degree of similarity between the two sequences and the length of the sequence match. For example, for a product score of 40, the match is accurate to within 1-2% error,
A product score of 70 is an exact match. Similar molecules are usually identified by selecting those molecules that exhibit a product score between 15 and 40, but lower scores identify them as related molecules.

The results of the Northern analysis are reported as a distribution of the percentage of libraries in which transcripts encoding LBAP occurred. The analysis includes the classification of cDNA libraries by organ / tissue and disease. Organ / tissue classifications include cardiovascular, dermal, developmental, endocrine, gastrointestinal, hematopoietic / immune, musculoskeletal, nervous, reproductive and urinary. Disease / symptom classifications include cancer, inflammation / trauma, cell proliferation, nerves and pooled. For each category, the libraries expressing the sequence of interest were counted and divided by the total number of libraries across categories. Table 3 shows the expression specific to each tissue and the ratio of expression specific to disease, disease or symptom.

5 Extension of Polynucleotide Encoding LBAP The full-length nucleic acid sequence of SEQ ID NO: 6-10 is generated by extending an appropriate fragment of the full-length molecule and designing oligonucleotide primers from this fragment. did. One primer was synthesized to initiate 5 'extension of the known fragment and the other primer was synthesized to initiate 3' extension of the known fragment. The starting primer is OLIGO 4.
Using software (National Biosciences) or other suitable program, is about 22-30 nucleotides in length and has a GC content of 50% or more;
The cDNA was designed to anneal to the target sequence at a temperature of about 68-72 ° C.
Hairpin structure and any extension of nucleotides resulting in primer-primer dimer was avoided.

This sequence was extended using a selected human cDNA library. If more than one step is necessary or desired, an additional or nested set of primers is designed.

High fidelity amplification was performed by PCR using methods well known to those skilled in the art. PCR is
Performed in a 96-well plate using a PTC-200 thermal cycler (MJ Research, Inc.). The reaction mixture contains a DNA template, 200 nmol of each primer, a reaction buffer containing Mg ²⁺ , (NH ₄ ) ₂ SO ₄ and β-mercaptoethanol, and Taq DNA polymerase (
Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies) and Pfu
DNA polymerase (Stratagene). Primer set PCI A and PCI B
The following parameters were used for Step 1 94 ° C for 3 minutes Step 2 94 ° C for 15 seconds Step 3 60 ° C for 1 minute Step 4 68 ° C for 2 minutes Step 5 Repeat steps 2, 3 and 4 20 times Step 6 68 ° C for 5 minutes Step 7 Store at 4 ° C. or use the following parameters for primer set T7 and SK +. Step 1 94 ° C for 3 minutes Step 2 94 ° C for 15 seconds Step 3 57 ° C for 1 minute Step 4 68 ° C for 2 minutes Step 5 Repeat steps 2, 3, and 4 20 times Step 6 68 ° C for 5 minutes Step 7 Store at 4 ° C. The DNA concentration in each well is determined using 0.5 μl of undiluted PCR product and 100 μl of PICOGREEN quantification reagent (0.25% (v / v) PICOGREEN; Molecular Probes, Eu) dissolved in 1 × TE.
gene OR) was distributed to each well of an opaque fluorometer plate (Coming Costar, Acton MA) and measured so that DNA could bind to the reagent. Put this plate in Fluor
Scan with oskan II (Labsystems Oy, Helsinki, Finland) to measure sample fluorescence and quantify DNA concentration. Aliquot 5-10 μl aliquots of the reaction mixture
Analyze by electrophoresis on a% agarose minigel to determine which reaction has successfully extended the sequence.

[0204] The extended nucleotides are desalted and concentrated, transferred to a 384-well plate, and subjected to CviJI choleravirus endonuclease (Molecular Biology Research, Madison WI).
And sonication or shearing was performed before religation to the pUC18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel, the fragments were excised, and the agar was digested with Agar ACE (Promega). The expanded clone is used for T4 ligase (New Eng
land Biolabs, Beverly MA) using the pUC 18 vector (Amersham Pharmacia Bio
tech) and treated with Pfu DNA polymerase (Stratagene) to fill in restriction site overhangs and transfected into competent E. coli cells. Transfected cells are selected in media containing antibiotics, individual colonies are selected and LB
The cells were cultured overnight at 37 ° C. in a 384-well plate of a / 2 × carb culture solution.

The cells were lysed and Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu
DNA was amplified by PCR using DNA polymerase (Stratagene) with the following parameters. Step 1 94 ° C for 3 minutes Step 2 94 ° C for 15 seconds Step 3 60 ° C for 1 minute Step 4 72 ° C for 2 minutes Step 5 Repeat steps 2, 3, and 4 29 times Step 6 72 ° C for 5 minutes Step 7 Store at 4 ° C. DNA was quantified with PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recovery were re-amplified under the above conditions. Dilute the sample with 20% dimethylsulphoxide (1: 2, v / v) and use DYENAMIC energy trans
fer sequencing primer and DYENAMIC DIRECT kit (Amersham Pharmacia
Biotech) or ABI PRISM BIGDYE Terminator cycle sequencing ready reac
The sequence was determined using the tion kit (Perkin-Elmer).

Similarly, using the nucleotide sequence of SEQ ID NO: 6-10, the above procedure, oligonucleotides designed for 5 ′ extension, and the appropriate genomic library to obtain 5 ′ regulatory sequences.

6 Labeling and Use of Individual Hybridization Probes Using the hybridization probes obtained from SEQ ID NOs: 6-10, cDNA
Genomic DNA or mRNA. Although the labeling of oligonucleotides of about 20 base pairs is specifically described, generally the same procedure is used for larger nucleotide fragments. Oligonucleotides can be transferred to OLIGO 4.06 software (National
Biosciences) and 50 pmol of each oligomer, 250 μCi of [γ- ³² P] adenosine triphosphate (Amersham Pharmacia Biot.
ech) and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotide is substantially purified using a SEPHADEX G-25 ultrafine molecular size exclusion dextran bead column (Amersham Pharmacia Biotech). Aliquots containing 10 ⁷ counts of labeled probe per minute were digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba1, or Pvu II (DuPont NEN). Used for typical membrane-based hybridization analysis of human genomic DNA.

DNA from each digest is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. The blot is washed sequentially at room temperature under increasingly stringent conditions up to 0.1 × sodium citrate saline and 0.5% sodium dodecyl sulfate to remove non-specific signals. Hybridization patterns are visualized and compared using autoradiography or alternative imaging means.

7 Array elements can be synthesized on the surface of a substrate by using a microarray chemical coupling procedure and an inkjet device. (For example, Baldeschweiler
, Supra). Also, using a predetermined array similar to dot blot or slot blot methods, utilizing thermal, UV, chemical or mechanical binding procedures,
Elements may be arranged and coupled to the surface of the substrate. Standard arrays can be manufactured by hand or using convenient methods and machines, and include any suitable number of elements. After hybridization, unhybridized probe is removed and the level and pattern of fluorescence are determined using a scanner. The degree of complementarity and relative abundance of each probe that hybridizes to an element on the microarray is evaluated by analyzing an image read by a scanner.

[0210] Full-length cDNAs, expressed sequence tags (ESTs), or fragments thereof, can include elements of a microarray. Suitable fragments for hybridization can be selected using software such as LASERGENE software (DNASTAR) well known to those skilled in the art. A full-length cDNA, EST, or a fragment thereof corresponding to one of the nucleotide sequences of the present invention, or a random selection from a cDNA library relevant to the present invention, is applied to a suitable substrate such as a glass slide. To place. By treating the cDNA thermally and chemically after, for example, UV crosslinking and further drying,
Fix to glass slides (eg, Schena, M. et al. (1995) Science 270: 467-47).
0; and Shalon, D. et al. (1996) Genome Res. 6: 639-645). A fluorescent probe is prepared and used for hybridization with an element on a substrate. The substrate is analyzed by the method described above.

8 Complementary polynucleotide The sequence complementary to the sequence encoding LBAP, or any portion thereof, may be replaced with a naturally occurring LBAP.
Used to detect and reduce, ie suppress, the expression of AP. Although the use of oligonucleotides containing about 15-30 base pairs is described, essentially the same method is used for smaller or larger sequence fragments. Design the appropriate oligonucleotides using OLIGO 4.06 software and the coding sequence of LBAP. To repress transcription, complementary oligonucleotides are designed from the most unique 5 'sequence and used to prevent promoter binding to the coding sequence. To suppress translation, complementary oligonucleotides are designed to prevent ribosome binding to the transcript encoding LBAP.

9. Expression of LBAP Expression and purification of LBAP is performed using a bacterial or virus based expression system. For expression of LBAP in bacteria, the cDNA is subcloned into an appropriate vector containing an antimicrobial resistance gene and an inducible promoter that increases the level of cDNA transcription. Examples of such promoters include, but are not limited to, the T5 or T7 bacteriophage promoter associated with the lac operator regulatory element and tr.
p-lac (tac) hybrid promoter. Recombinant vector, BL21 (D
Transform into a suitable bacterial host such as E3). Antibiotic resistant bacteria express LBAP by induction with isopropyl β-D thiogalactopyranoside (IPTG). Expression of LBAP in eukaryotic cells has been demonstrated in insect or mammalian cell lines by using Autographica californica nuclear polyhedrosis virus (commonly known as baculovirus).
AcMNPV). The non-essential polyhedrin gene of the baculovirus is replaced with the cDNA encoding LBAP by either homologous recombination or bacterial-mediated gene transfer involving a transfer plasmid intermediate. Virus infectivity is maintained and strong polyhedrin promoters drive high levels of cDNA transcription. In most cases, the recombinant baculovirus is Spodoptera frugiperda (Sf9)
It is used to infect insect cells, and in some cases, to infect human hepatocytes. The latter infection requires additional genetic alterations to the baculovirus. (For example, Engelhard. EK et al. (1994) Proc. Natl. Acad. Sci. USA 91: 322.
4-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945).

In most expression systems, LBAP is, for example, glutathione S-transferase (G
ST), or synthesized as a fusion protein with a peptide epitope tag such as FLAG or 6-His, which allows for rapid, one-step affinity-based purification of the recombinant fusion protein from crude cell lysates. GST, a 26 kilodalton enzyme from Schistosoma japonicum, allows the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amer
sham Pharmacia Biotech). After purification, LBAP to GST at specific operating sites
The part can be proteolytically cut. FLAG, an eight amino acid peptide
Allows immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, allows for purification on metal chelating resins (QIAGEN). Ausubel (1995, earlier ) describes methods for protein expression and purification.
Id , ch 10 and 16). Purified LBAP obtained by these methods
Can be used directly in the following assays.

10 Demonstration of LBAP Activity Assays for LBAP activity measure LBAP expression on the cell surface.
The cDNA encoding LBAP is transfected into a mammalian (non-human) cell line. Cell surface proteins are labeled with biotin as in de la Fuente, MA et al. (1997, Blood 90: 2398-2405). Immunoprecipitation is performed using an antibody specific for LBAP, and the immunoprecipitated sample is analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Unlabeled immunoprecipitate (immunoprecipita
The ratio of labeled immunoprecipitate to nt) is based on LBAP expressed on the cell surface.
Is proportional to the amount of

11 Functional Assays The function of LBAP was measured at physiologically elevated levels of LB in mammalian cell culture systems.
Assay by expression of the sequence encoding AP. The cDNA is subcloned into a mammalian expression vector containing a strong promoter that expresses the cDNA at high levels.
Selected vectors include pCMV SPORT plasmid (Life Technologie) and pCR3
.1 plasmids (Invitrogen), each of which contains the cytomegalovirus promoter. 5-10 μg of the recombinant vector is transfected instantaneously, for example, into an endothelial or hematopoietic cell line by liposome preparation or electroporation. Also, 1-2 μg of additional plasmid containing the sequence encoding the labeled protein is co-transfected. Expression of the labeled protein allows one to distinguish between transfected and non-transfected cells and to reliably predict expression of cDNA from a recombinant vector. Selected fluorescent proteins include green fluorescent protein (GF
P; Clontech), CD64, or CD64-GFP fusion protein. Using flow cytometry (FCM), a technology based on automated laser optics, GFP
Alternatively, transfected cells expressing CD64-GFP are identified, and the properties of the cells (eg, state of apoptosis) are assessed. FCM detects and quantifies the uptake of fluorescent molecules that diagnose the phenomenon of preceding or simultaneous cell death. These phenomena include changes in nuclear DNA content as measured by staining DNA with propidium iodide,
Down-regulation of DNA synthesis as measured by reduced uptake of bromodeoxyuridine, altered cell surface and intracellular protein expression as measured by reactivity with specific antibodies, and cells with fluorescein-bound annexin V protein Includes changes in plasma membrane composition as measured by binding to the surface. For flow cytometry methods, see Ormerod, MG (1994) Flow Cytometry, Oxford, Ne.
w York, NY.

The effect of LBAP on gene expression was determined by the sequence encoding LBAP as well as CD64 or
It can be assessed using highly purified cell populations transfected with any of the CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transformed cells and bind to conserved regions of human immunoglobulin G (IgG). Transformed cells can be separated from non-transformed cells using magnetic beads coated with either human IgG or CD64 antibodies (DYNAL, Lake Success, NY). mRN
A can be purified from cells by methods well known to those skilled in the art. Expression of mRNA encoding LBAP and other genes of interest can be analyzed by Northern analysis or microarray technology.

12 Production of LBAP-Specific Antibodies Polyacrylamide gel electrophoresis (PAGE; see, eg, Harrington, MG (1990
Rabbits are immunized according to standard protocols to generate antibodies with LBAP substantially purified using Methods Enzymol. 182: 488-495) or other purification techniques.

Alternatively, the amino acid sequence of LBAP is analyzed using LASERGENE software (DNASTAR) to determine a region having high immunogenicity, and a corresponding oligopolypeptide is synthesized, and is used by those skilled in the art. The antibody is produced by the method. Details of how to select suitable epitopes, such as those near the C-terminus or within the hydrophilic region, are described in the literature of those skilled in the art (see, for example, Ausubel, 1995, supra , ch. 11).

Typically, an oligopeptide 15 residues in length is converted to ABI 431 using the chemistry of the fmoc method.
A Peptide Synthesizer (Perkin-Elmer) was used for synthesis and MBS (N-maleimidoben)
The reaction using zoyl-N-hydroxysuccinimide ester) resulted in KLH (Sigma, St. Louis,
MO) to enhance immunogenicity (see, eg, Ausubel, supra ). Rabbits are immunized with the oligopeptide-KLH complex in Freund's complete adjuvant. The resulting antiserum is, for example, a peptide bound to plastic, 1%
Tested for anti-peptide activity by blocking with BSA, reacting with rabbit antiserum, washing and reacting with radioiodine-labeled goat anti-rabbit IgG.

13 Purification of Natural LBAP Using Specific Antibodies Immunoaffinity chromatography using antibodies specific for LBAP
The natural or recombinant LBAP is substantially purified. Immunoaffinity column is LB
AP antibodies are made by covalently attaching to an activated chromatography resin such as CNBr-activated Sepharose (Amersham Pharmacia Biotech).
After bonding, block and wash the resin according to the manufacturer's instructions.

The culture solution containing LBAP is passed through an immunoaffinity column, and the column is washed under conditions that preferentially adsorb LBAP (eg, a high ionic strength buffer in the presence of a surfactant). Conditions that disrupt antibody / LBAP binding (eg, pH2
The column is eluted with ~ 3 buffers or a high concentration of chaotrope such as urea or thiocyanate ions to recover LBAP.

14 Identification of molecules that interact with LBAP Label LBAP or a biologically active fragment thereof with ¹²⁵ I Bolton-Hunter reagent (eg, Bolton AE and WM Hunter (1973) Biochem. 133: 529-
539). Candidate molecules previously placed in the wells of a multiwell plate are labeled L
Incubate with BAP, wash and assay any wells with labeled LBAP complex. Using the data obtained at different LBAP concentrations, values for the binding, affinity and number of LBAP with the candidate molecule are calculated.

Alternatively, molecules that interact with LBAP are identified in Fields, S. and O. Song (1989, Natu
re 340: 245-246) yeast two-hybrid system and the MATCHMAKER system (Clon
tech), using a commercially available kit based on a two-hybrid system.

One skilled in the art will be able to make various modifications of the methods and systems described herein without departing from the scope and spirit of the invention. Although the invention has been described with respect to particular preferred embodiments, it is to be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims.

[0225] BRIEF DESCRIPTION Table 1 Table, ID number (SEQ ID NO) of the polypeptide sequence and nucleotide sequence used to construct the full length sequence encoding LBAP, clone identification number (Clone ID) , A cDNA library, and a cDNA fragment.

Table 2 shows the characteristics of each polypeptide sequence, including potential motifs and homologous sequences, and the methods, algorithms and searchable databases used to identify LBAP.

Table 3 shows the tissue-specific expression patterns of each nucleic acid sequence determined by Northern analysis, the diseases, disorders or conditions associated with these tissues, and the vectors into which each DNA was cloned.

Table 4 shows tissues used for preparing a cDNA library from which a cDNA clone encoding LBAP was isolated.

[0229] Table 5 shows the tools, programs and algorithms used for the analysis of LBAP, their descriptions, references and parameter thresholds.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/00 C07K 14/47 4C084 37/00 16/18 4H045 C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/68 A 5/10 G01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/68 C12N 15/00 ZNAA G01N 33/15 5/00 A 33/50 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, W, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, 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, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Hillman, Jennifer El California, USA 94040 Mountain View # 12 Monrodra Eve 230 (72) Inventor Ryu, Dung Aina M. 95136, San Jose Park, Belmont Place, California, USA 55 (72) Inventor Bogun, Mariah Earl San Leandro Santiago Road, 94577, California, USA 14244 (72) Inventor Tongue, Wye Tom 95118, California, United States San Jose Runwick Court 4230 (72) Inventor Azimsay, Yarda 94545, California, United States Hayward Rock Springs Drive 2045 F-term (reference) 2G045 AA34 AA35 CB01 DA13 DA36 FB02 FB05 FB08 FB12 4B024 AA01 AA11 BA44 CA01 GA11 GA27 HA12 4B063 QA01 QA18 QA19 QQ03 QQ42 QQ53 QQ61 QR32. ZA511 ZA512 ZB011 ZB012 ZB111 ZB1 12 ZB261 ZB262 4H045 AA10 AA11 BA10 CA42 DA76 EA23 EA51 FA74

Claims (23)

[Claims] 1. An amino acid sequence selected from the group consisting of: (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5; (C) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, and (d) SEQ ID NO: An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: an immunogenic fragment of an amino acid sequence selected from the group consisting of 1-5. 2. The isolated polypeptide of claim 1, wherein the polypeptide is selected from the group consisting of SEQ ID NOs: 1-5. 3. An isolated polynucleotide encoding the polypeptide of claim 1. 4. The isolated polynucleotide of claim 3, wherein the isolated polynucleotide is selected from the group consisting of SEQ ID NO: 6-10. 5. A recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide of claim 3. 6. A cell transformed with the recombinant polynucleotide of claim 5. 7. A genetic transformant comprising the recombinant polynucleotide according to claim 5. 8. The method for producing the polypeptide according to claim 1, wherein (a) culturing the cell under conditions suitable for expression of the polypeptide, wherein the cell comprises: Culturing, transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide encoding the polypeptide of (b), and (b) recovering the polypeptide so expressed A method for producing a polypeptide, characterized in that: 9. An isolated antibody that specifically binds to the polypeptide of claim 1. 10. A polynucleotide sequence selected from the group consisting of (a) SEQ ID NO: 6-10 and (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 6-10. A naturally occurring polynucleotide sequence having at least 70% sequence identity, (c) a polynucleotide sequence complementary to (a), and (d) a polynucleotide sequence complementary to (b); (E) an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of: RNA equivalents of (a) to (d). 11. An isolated polynucleotide comprising at least 60 contiguous nucleotides of the polynucleotide of claim 10. 12. A method for detecting a target polynucleotide comprising the polynucleotide sequence of claim 10 in a sample, the method comprising: (a) synthesizing the sample to complement the target polynucleotide in the sample; In the process of hybridizing with a probe comprising at least 16 contiguous nucleotides containing a unique sequence, under the conditions that a hybridization complex is formed between the probe and the target polynucleotide, Specifically hybridizes with the target polynucleotide, and (b) detecting whether or not the hybridization complex is present, and if present, optionally determining the amount thereof. And a detecting step. 13. The method of claim 12, wherein said probe comprises at least 30 contiguous nucleotides. 14. The method of claim 12, wherein said probe comprises at least 60 contiguous nucleotides. 15. A pharmaceutical composition comprising an effective amount of the polypeptide of claim 1 and a pharmaceutically acceptable excipient. 16. A method of treating a disease or condition associated with reduced expression of functional LBAP, comprising administering to the patient in need of such treatment the pharmaceutical composition of claim 15. . 17. A method for screening a compound effective as an agonist of the polypeptide of claim 1, comprising: (a) exposing a sample containing the polypeptide of claim 1 to the compound; and (b) Detecting the activity of the agonist in the sample. 18. A pharmaceutical composition comprising an agonist compound identified by the method of claim 17 and a pharmaceutically acceptable excipient. 19. A method of treating a disease or condition associated with reduced expression of functional LBAP, comprising administering a pharmaceutical composition of claim 18 to a patient in need of such treatment. . 20. A method for screening a compound effective as an antagonist of the polypeptide of claim 1, comprising: (a) exposing a sample containing the polypeptide of claim 1 to the compound; and (b) Detecting the activity of the antagonist in the sample. 21. A pharmaceutical composition comprising an antagonist compound identified by the method of claim 20 and a pharmaceutically acceptable excipient. 22. A method of treating a disease or condition associated with overexpression of functional LBAP, comprising the step of administering the pharmaceutical composition of claim 21 to a patient in need of such treatment. 23. A method for screening a compound effective for altering expression of a target polypeptide comprising the sequence of claim 4, comprising: (a) exposing a sample comprising said target polypeptide to said compound. A screening method, comprising: (b) detecting a change in expression of the target polypeptide.