JP2002530080A - Immunoglobulin / Superfamily / Protein - Google Patents

Abstract

  (57) [Summary] The present invention provides human immunoglobulin superfamily proteins (IGFAM) and polynucleotides that identify and encode IGFAM. 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 IGFAM.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field The present invention relates to nucleic acid and amino acid sequences of the immunoglobulin superfamily proteins, as well as cancer, the diagnosis of immune system abnormalities and infections, it relates to uses of these sequences in the treatment and prevention.

BACKGROUND all vertebrate invention, viruses, bacteria, have developed sophisticated and complex immune system to defend itself from infection of fungi and parasites. This defense system is mediated by cell surface molecules and soluble molecules that function in recognition, adhesion or binding.
The vertebrate immune system has evolved from a common evolutionary precursor (ie, these proteins have structural homology). Many molecules outside of the immune system with similar functions are also derived from this same evolutionary precursor.

[0003] An important feature of the immune system is its ability to recognize and destroy foreign molecules or antigens.
Antigen recognition is primarily mediated by secreted and transmembrane proteins expressed by leukocytes such as granulocytes, monocytes and lymphocytes. Most of these proteins belong to the immunoglobulin (Ig) superfamily. Cell surfaces and soluble molecules of the immune system are Igs containing one or more repeats of a conserved structural Ig domain.
It is classified into each member of the super family. Ig domains with a length of 70-110 amino acid residues are homologous to either the Ig variable-like (V) domain or the Ig constant-like (C) domain. The Ig domains were shown as antiparallel β-sheets linked by disulfide bonds in a structure called an immunoglobulin fold.
Members of the Ig superfamily include antibodies (Abs), T cell receptors (TCRs), and
MHC class I and II proteins, CD2, CD3, CD4, CD8, poly Ig receptor, Fc receptor,
Includes nerve cell adhesion molecule (NCAM) and platelet-derived growth factor (PDGFR).

[0004] Ig domains (V and C) are regions of conserved amino acid residues that make a polypeptide a globular tertiary structure. This globular tertiary structure is called an immunoglobulin (or antibody) fold and consists of two generally parallel layers of β-sheets. Conserved cysteine residues are 55-75 amino acid residues in length and form an intrachain disulfide-bonded loop connecting the two β-sheet layers. Due to the hydrophobic and hydrophilic interaction of amino acid residues in the β-chain, the immunoglobulin fold (amino acid residues facing the inside of the chain are hydrophobic and amino acid residues facing the outside are hydrophilic ) Is stabilized. The V domain consists of a polypeptide longer than the C domain and has an additional pair of β chains within the immunoglobulin fold.

A common feature of Ig superfamily genes is that each sequence in the Ig domain is encoded by one exon. This superfamily, evolved from a gene encoding one Ig domain, can be thought to be involved in mediating cell-cell interactions. New members of the superfamily are born by exon and gene duplication. New Ig superfamily proteins have varying numbers of V and / or C domains. Another evolutionary feature of this superfamily is undergoing DNA rearrangement, a unique feature that maintains the antigen receptor members of this family.

[0006] Many members of the Ig superfamily are integral cell membrane proteins with extracellular Ig domains. These transmembrane domains and hydrophobic amino acid residues in the cytoplasmic tail are highly diverse, with zero or very low homology between Ig family members or to known signaling structures. There are exceptions to this general superfamily description. For example, the cytoplasmic tail of PDGFR has tyrosine kinase activity. In addition, Thy-1 is a glycoprotein found on thymocytes and T cells. This protein does not have a cytoplasmic tail, but instead is attached to the cell membrane by a covalent glycophosphatidylinositol bond.

[0007] Another common feature of many Ig superfamily proteins is the interaction between Ig domains that is essential for the function of these molecules. The interaction between the Ig domains of the multimeric protein may be homophilic or heterophilic (ie, the same or different Ig
Domain). Antibodies are multimeric proteins that have both homophilic and heterophilic interactions between Ig domains. The pairing between the constant regions of the H chains forms the Fc region of the antibody, and the pairing between the variable regions of the L and H chains forms the antigen-binding site of the antibody. Heterogeneous interactions also occur between the Ig domains of different molecules. Immune system, adhesion between molecules in significant intermolecular interactions developmental and maturation nervous system, due to these interactions (Abbas, AK et al. ( 1991) Ce llular and Molecular Immunology, WB Saunders Company, Philadelphia, PA
, pp. 142-145).

Antibodies Antibodies or immunoglobulins constitute a member of the Ig superfamily and are a major component of the humoral immune response. Antibodies are expressed on the surface of B cells or
It is either secreted into the blood by cells. Antibodies bind and render harmless foreign antigens carried by the blood. A prototype antibody is a tetramer consisting of two identical polypeptide heavy chains (H chains) and two identical polypeptide light chains (L chains) linked together by disulfide bonds. This configuration results in a characteristic Y-shaped antibody molecule. The five antibody classes, IgA, IgD, IgE, IgG, and IgM, are determined by the α, δ, ε, γ, and μ chains, respectively. Κ and λ for L chain
One of which can form a pair with any pair of H chains. The most common antibody class IgG found in blood is tetramer, while other antibody classes are usually multimers or variants of this basic structure.

[0009] The heavy and light chains each have an N-terminal variable region and a C-terminal constant region. The constant region is comprised of about 110 amino acids of the light chain and about 330-440 amino acids of the heavy chain. The amino acid sequences of the constant regions are almost identical between H chains or L chains of a particular class. The variable region is comprised of about 110 amino acids in both the heavy and light chains.
However, the amino acid sequences of the variable regions differ between certain classes of H or L chains. Within each variable region of the H or L chain, there are three hypervariable regions of about 5 to 10 amino acids each having a wide variety of sequences. Antibody molecules combine the hypervariable regions of the H and L chains to form an antigen recognition site (Alberts. 13. et al. (1994) Molecular Biology of the Cell , Garland Publ.
ishing, New York, NY. See pages 1206-1213 and 1216-1217).

[0010] Each of the heavy and light chains has a repeating Ig domain. For example, a typical heavy chain is 4
Three Ig domains, three of which are in the constant region and one of which is in the variable region, which contributes to the formation of an antigen recognition site. Similarly, a typical light chain has two I
It has a g domain, one in the constant region and one in the variable region. Further, it is known that H chains such as μ associate with other polypeptides during B cell differentiation. One such polypeptide, called 8HS-20, is itself a member of the Ig superfamily and has one Ig domain (Shirasawa, T.
et al. (1993) EMBOJ. 12: 1827-1834).

[0011] The immune system recognizes and responds to any foreign molecule that has entered the body. Therefore, the immune system must have all kinds of antibodies against all potential antigens. Such antibody diversity results from somatic recombination of the gene segments encoding the variable and constant regions. These gene segments are linked by site-specific recombination that occurs between highly conserved DNA sequences adjacent to each gene segment. Due to the presence of hundreds of different gene segments, the combination can produce millions of different genes. Also, incorrect antibody binding and abnormally high somatic mutations in these segments lead to further antibody diversification.

[0012] Antibodies can also be described from two main functional domains. Antibody recognition is mediated by the Fab (antigen-binding fragment) region of the antibody, and effector functions are mediated by the Fc (crystallizable fragment) region. When the antibody binds to an antigen, such as a bacterium, phagocytic white blood cells, such as macrophages and neutrophils, begin to destroy the antigen. These cells express a cell surface receptor that specifically binds to the antibody Fc region, whereby phagocytic cells take up, digest, and degrade the antibody-bound antigen. The Fc receptor expressed by phagocytes is a single transmembrane glycoprotein consisting of about 300-400 amino acids (Sears, DW et al. (1990) 3. Immunol. 144
: 371-378). The extracellular portion of the Fc receptor usually has two or three Ig domains.

[0013] Fc receptor-specific variants have been identified in bone marrow cells and lymphocytes (Samaridis. 3
and Colonna. M. (1997) Eur. 3. Immunol. 27: 660-665). Like typical Fc receptors, these proteins have extracellular Ig domains and are encoded by cDNAs called ILT1 and ILT2 (Ig-like transcripts 1 and 2). However, the transmembrane and cytoplasmic domains are quite diverse. In particular, the cytoplasmic domain is extensive and has protein motifs consistent with a role for intracellular signaling.

[0014] New members of the Ig superfamily appear to play a structural role in controlling monocyte migration from epithelial or endothelial cells to sites of inflammation. This protein, called the junctional adhesion molecule (JAM), is located at the tight junction between epithelial and endothelial cells (Martin-Padura, I. et al.
1998) J. Cell Biol. 142: 117-127). JAM is 300 amino acids long, 2
It has one Ig domain. When a monoclonal antibody (mAb) is sent to JAM,
The migration of monocytes into the itro endothelial cell layer was inhibited. Furthermore, systemic administration of this mAb to mice prevented recruitment of monocytes to inflammatory sites.

There is also a description of a viral protein having an Ig domain (Senkevich, T
G. et al. (1996) Science 273: 813-816). These proteins include the MHC-like homologs identified in the human oncogenic poxvirus Molluscum contasiosum . Such proteins may provide a mechanism by which the virus can escape the host immune surveillance system.

T cell receptors are structurally and functionally related to antibodies (Alberts, supra, pp. 12
28-1229). T cell receptors are cell surface proteins that bind foreign antigens and mediate the immune response at various points. A typical T cell receptor is a heterodimer consisting of two disulfide-linked polypeptide chains called the α and β chains. Each chain is about 280 amino acids in length and has one variable region and one constant region. Each of the variable and constant regions is folded into an Ig domain. The variable regions of the α and β chains form a heterodimer and serve as an antigen recognition site. Diversity in T cell receptors results from somatic recombination of gene segments encoding the α and β chains. T cell receptors recognize small peptide antigens that appear on the surface of antigen presenting cells and pathogen infected cells. These peptide antigens are presented on the cell surface by MHC proteins that provide the correct context for antigen recognition.

A specific cell connection may occur at a portion where the subsynaptic membrane glycoprotein cells come into contact with each other. These cell junctions include transduction junctions that mediate pathways of chemical and electrical signals between cells.
Transmission connections between neurons in the central nervous system are known as synaptic connections. They consist of the precursors of synaptic neurons and the membrane and cytoskeleton of mature synaptic neurons. Subsynaptic membrane (SM) and postsynaptic density (PSD: postsynaptic de
Certain glycoproteins found in biochemically isolated synaptic subfractions, such as nsity fragments, have been identified and their functions elucidated. One of them is SM glycoprotein
gp50 has been identified as the β2 subunit of the Na ⁺ / K ⁺ ATase.

[0018] The SM and PSD glycoproteins with oligosaccharide domains facing synaptic junctions are conveniently located for mediating adhesion interactions between neurons. PS
The D component, PAC1 glycoprotein, has been identified as a member of the cadherin family, a protein involved in Ca2 ⁺ -dependent cell-cell adhesion in vertebrate tissues. These molecular-mediated adhesion interactions are also aided by the presence of integrin-type adhesion molecules, members of the SM superfamily of SM, and NACM.

The two glycoproteins gp65 and gp55 are the major components of the subsynaptic membrane from rat forebrain. They are members of the Ig superfamily with three and two Ig domains, respectively. Being a member of the Ig superfamily, it has been proposed that these potential functions include mediation of adhesive interactions at synaptic junctions (Langnase, K. et al. (1997) J. Biol. Chem. 272 (2): 821-827
).

The discovery of novel immunoglobulin superfamily proteins and polynucleotides encoding them provides novel compositions useful for the diagnosis, prevention and treatment of cancer, immune system diseases, and infectious diseases. Can meet the needs of the field.

SUMMARY OF THE INVENTION The present invention collectively comprises "IGFAM", "IGFAM-1", "IGFAM-2", "IGFAM-3", and "IGFAM-3".
GFAM-4, IGFAM-5, IGFAM-6, IGFAM-7, IGFAM-8, IGFAM-9, IGFAM-1
0, IGFAM-11, IGFAM-12, IGFAM-13, IGFAM-14, IGFAM-15, IGFAM-1
6 "," IGFAM-17 "," IGFAM-18 "and" IGFAM-19 "characterized by a substantially purified polypeptide, the immunoglobulin superfamily protein. In one embodiment, the present invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-19 and fragments thereof. The present invention also includes a polypeptide comprising an amino acid sequence in which one or more conservative amino acids have been substituted from an amino acid sequence selected from the group consisting of SEQ ID NO: 1-19.

The present invention also provides a substantially purified amino acid sequence having at least 90% or more amino acid identity to at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-19 and fragments thereof. Provide mutants. The invention further provides an isolated and purified polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19 and fragments thereof. In addition, the present invention provides SEQ ID
NO: 1-19 and an isolated and purified polypeptide having at least 90% or more polynucleotide sequence identity to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: Mutated nucleotide sequences are included.

In addition, the present invention provides an isolation which hybridizes under stringent conditions to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-19 and fragments thereof. To provide a purified and purified polynucleotide. The present invention also provides an isolated and purified polynucleotide having a sequence complementary to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19 and fragments thereof. I will provide a.

The present invention also provides a method for detecting a polynucleotide in a sample containing a nucleic acid, the method comprising the steps of: (a) hybridizing at least one polynucleotide of a sample with a complementary sequence of the polynucleotide; Forming a hybridization complex, and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding a polypeptide in the biological sample. And a detection process having a characteristic. In one embodiment of the invention, the method further comprises amplifying the polynucleotide prior to hybridization.

Further, the present invention provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 20-38 and fragments thereof. The present invention also relates to an isolated and purified polynucleotide having at least 90% or more polynucleotide sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 20-38 and fragments thereof. Mutant sequences are provided. Further, the present invention provides an isolated and purified polynucleotide having a sequence complementary to a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 20-38 and fragments thereof.

Further, the present invention provides an expression vector comprising at least a fragment of a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-19. In another embodiment, the expression vector is contained in a host cell.

The present invention also provides a method for producing a polypeptide, the method comprising: (a) a host cell comprising an expression vector containing the polynucleotide of the present invention under conditions suitable for expression of the polypeptide; And (b) recovering the polypeptide from the medium of the host cell.

Further, the present invention provides a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-19 and fragments thereof together with a suitable pharmaceutical carrier. provide.

Further, the present invention includes a purified antibody that binds to a polypeptide selected from the group consisting of SEQ ID NO: 1-19 and fragments thereof. The present invention also provides a purified agonist and a purified antagonist of the polypeptide.

Further, the present invention provides a method of treating or preventing a disease associated with a decrease in the expression or activity of IGFAM, the method comprising the steps of:
NO: 1-19 and a step of administering an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of a fragment thereof and a suitable pharmaceutical carrier. .

Further, the present invention provides a method of treating or preventing a disease associated with increased expression or activity of IGFAM, the method comprising the steps of providing a subject in need of such treatment with SEQ ID NO:
And administering an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of NO: 1-19 and fragments thereof.

[0032] 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.

As used herein, 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.

[0034] 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 in the present invention are cited to describe and disclose 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 "IGFAM" is derived from any species, particularly mammalian species, including cows, sheep, pigs, mice, horses, and humans, and is natural, synthetic, semi-synthetic, or Refers to the amino acid sequence of substantially purified IGFAM obtained from recombinant sources.

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

The term “allelic variant” refers to another form of the gene encoding IGFAM. An allelic variant has a mutated mRN due to at least one mutation in the nucleic acid sequence.
A or polypeptide occurs, but their structure or function may or may not change. Genes without naturally occurring allelic variants, 1
There is one that exists or many that 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 IGFAM includes a variety of nucleotides that result in a polynucleotide encoding the same IGFAM or a polypeptide comprising at least one functional characteristic of IGFAM. And those sequences with deletions, insertions or substitutions of This definition includes improper or unexpected hybridization of the polynucleotide sequence encoding IGFAM to an allelic variant at a position different from the normal chromosomal location, or specific oligonucleotide probes of the polynucleotide encoding IGFAM. Includes polymorphisms that are easily detectable or difficult to detect. The encoded protein may also be "mutated" and may contain substitutions, deletions or insertions of amino acid residues that result in a silent change and result in a functionally equivalent IGFAM. Intentional amino acid substitutions may lead to similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues, as long as the biological or immunological activity of IGFAM is retained. May 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 naturally occurring 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 form of the amino acid sequence associated with the described protein molecule. It is not limited to the amino acid sequence.

[0040] The term "amplification" refers to the production of additional copies of a nucleic acid sequence, usually performed using polymerase chain reaction (PCR) techniques well known to those skilled in the art.

The term “antagonist” is a molecule that inhibits or attenuates the biological activity of IGFAM. Antagonists include proteins, such as antibodies, nucleic acids, carbohydrates, small molecules or any other compounds or compositions, which either directly interact with IGFAM or are components of a biological pathway in which IGFAM is involved. Regulates the activity of IGFAM.

The term “antibody” refers to whole molecules, Fa, F (ab ′) ₂ , and Fv that can bind antigenic determinants.
Refers to those fragments, such as fragments. Antibodies that bind to IGFAM polypeptides can be prepared using untreated polypeptides or using fragments thereof containing small peptides involved as immunizing antigens. 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 bind chemically to the peptide include 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 component that contains a nucleic acid sequence that is complementary to the “sense” strand of a particular nucleic acid sequence. Antisense molecules can be produced by any method, including synthesis and transcription. This complementary nucleotide, once introduced into the cell,
Combines with natural sequences produced by cells to form duplexes, preventing transcription and translation. The expression “negative” or “minus (−)” may refer to the antisense strand, 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 a natural immunoreaction that elicits a specific immune response in the appropriate animal or cell and binds to a specific antibody.
Refers to the ability of IGFAM, recombinant IGFAM or synthetic IGFAM 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 comprising a polynucleotide encoding IGFAM or a fragment of IGFAM 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 in which unnecessary bases have been separated by resequencing or a 5 ′ direction and / or 3 ′ position using an XL-PCR kit (The Perkin Elmer Corp., Norwalk, Conn.). 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, in some cases, one or more duplicated sequences of a publicly available EST or cDNA. 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 naturally occurring 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 IGFAM or a polynucleotide encoding IGFAM 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, some fragments contain 5-100
Zero consecutive nucleotides or amino acid residues may be included. Probes, primers, antigens, therapeutic molecules or fragments used for other purposes are at least 5, 10
, 15, 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 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: 20-38 include, for example, regions of a unique polynucleotide sequence that specifically identify SEQ ID NO: 20-38, which differ from other sequences in the same genome. Is included. Certain fragments of SEQ ID NO: 20-38 are useful, for example, in hybridization and amplification techniques, and similar methods that distinguish SEQ ID NO: 20-38 from related polynucleotide sequences. A fragment of SEQ ID NO: 20-38 or SEQ I
The exact length of the region of D NO: 20-38 can be routinely determined by known techniques based on the purpose of the fragment.

Certain fragments of SEQ ID NO: 1-19 are encoded by certain fragments of SEQ ID NO: 20-38. Certain fragments of SEQ ID NO: 1-19 include regions of a unique amino acid sequence that specifically identify SEQ ID NO: 1-19. For example, one fragment of SEQ ID NO: 1-19
It is useful as an immunogenic peptide for the production of an antibody that specifically recognizes ID NO: 1-19. The exact length of a fragment of SEQ ID NO: 1-19 or a region of SEQ ID NO: 1-19 that corresponds to a fragment can be routinely determined by known techniques based on the purpose of the fragment.

The term “similarity” means 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 term “percent identity” or “percent identity” for a polynucleotide sequence refers 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.

[0059] Despite nucleic acid sequences that do not show high identity, the genetic code may encode similar amino acid sequences due to degeneracy. 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 such that the antibody retains its original binding ability and is closer to a human antibody.

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 wash 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 bonding 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 “microarray” refers to an arrangement of various polynucleotides on a substrate.

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

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

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-gene (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 IGFAM, 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) which are
The target polynucleotide can be annealed 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.

Methods for preparing and using probes and primers are described, for example, in 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 to be used as primers are selected by a computer program 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.
g 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 Huma
n The Genome Mapping Project Resource Center, available from Cambridge UK), hybridizes to either the most conserved or the least conserved regions of the aligned nucleic acid sequences to design primers based on multiple sequence alignments Primers can be selected. 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 natural sequence, but a sequence that is an artificial combination of two or more different, spaced apart segments of sequence. This artificial combination is often performed by chemical synthesis, but more generally, for example, as described in Sambrook, supra.
The isolated segments of nucleic acid are artificially manipulated by genetic engineering techniques as described in ( supra ). The “recombinant nucleic acid” also includes a nucleic acid that is changed simply by adding, substituting or deleting a part of the nucleic acid. 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 is part of a vaccinia virus-based viral vector that elicits a protective immune response in the mammal. Can be

The term “sample” is used in its broadest sense. Nucleic acids encoding IGFAM or fragments thereof, or samples suspected of containing IGFAM itself, include body fluids,
Chromosomes isolated from cells, extracts from organelles or cell membranes, and cells, genomic DNA, RNA or cDNA in solution, or bound to a solid support, tissues, tissue prints, etc. .

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% or more, preferably about 75% or more, and most preferably 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 the 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.

A “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. .

Invention The present invention relates to a novel human immunoglobulin superfamily protein (IG
FAM), polynucleotides encoding IGFAM, and the use of these compositions for the diagnosis, treatment or prevention of cancer, immune system abnormalities and infectious diseases.

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

Each column of Table 2 shows various properties of each polypeptide of the present invention.
EQ ID NO, number of amino acid residues in each polypeptide in column 2, potential phosphorylation sites in column 3, potential glycosylation sites in column 4, signature sequence in column 5 (signa
Amino acid residues containing motifs) and motifs, the sixth row is a GenBank homolog identified by BLAST analysis, Is shown. Each polypeptide was characterized by sequence homology and protein motifs using the methods in column 7. In the fifth column, all but one of the polypeptides of the invention have one or more Ig domains predicted by a protein function analysis program such as PROFILESCAN, BLIMPS, PFAM and MOTIFS. Note that it is included. However, polypeptides of the invention lacking the predicted Ig domain (SEQ ID NO:
4) shows significant similarity to the viral Ig-containing protein MC51L-53L-54L.

Each column of Table 3 shows a disease, disorder or condition related to the tissue specificity as well as the nucleotide sequence encoding IGFAM. The first column of Table 3 shows the nucleotide SEQ ID NO. The second column shows a fragment of the nucleotide sequence of the first column. These fragments are, for example,
SEQ ID NO: 20-38 or to identify the related polynucleotide sequence and SEQ ID NO:
Useful in hybridization or amplification techniques to distinguish between D NO: 20-38. The polypeptides encoded by these fragments are useful, for example, as immunogenic peptides. The third column shows the tissue classification as a percentage of all tissues expressing IGFAM. Column 4 shows the percentage of all tissues expressing IGFAM of the disease, disorder or condition associated with those tissues. Column 5 shows the vector used for subcloning the cDNA library. Note that the nucleotide sequences of SEQ ID NOs: 23 and 27 are mainly expressed in cells and tissues involved in the hematopoietic system, immune system and inflammation.

Each column of Table 4 provides a description of the tissue used to create a cDNA library from which a cDNA clone encoding IGFAM is isolated. The first row is the nucleotide SEQ ID NO,
Row 2 is the cDNA library from which these clones were isolated, and row 3 is the cDNA library from row 2.
The tissue source and other descriptive information relevant to the DNA library are indicated, respectively.

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

[0097] The invention further includes polynucleotides encoding IGFAM. In certain embodiments, the invention includes a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 20-38 encoding IGFAM.

Further, the present invention includes a mutant sequence of a polynucleotide encoding IGFAM. In particular, a variant sequence of such a polynucleotide may have at least about 80% or more, or at least about 90% or more, or even at least about 95% or more polynucleotide sequence identity to the polynucleotide sequence encoding IGFAM. Have. Certain embodiments of the present invention include a variant sequence of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 20-38, which is selected from the group consisting of SEQ ID NOs: 20-38. At least about 80% or more, or at least about 90% or even at least about 95% or more polynucleotide sequence identity to the nucleic acid sequence. Any of the above mutant sequences of the polynucleotide may have at least one of the functional or structural characteristics of IGFAM.
It is possible to encode an amino acid sequence containing

The degeneracy of the genetic code results in the production of a large number of IGFAM-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 made according to the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring IGFAM, and all such variations are to be considered specifically disclosed herein.

Preferably, the nucleotide sequence encoding IGFAM and its mutant sequences are capable of hybridizing, under appropriately selected stringency conditions, to the nucleotide sequence of naturally occurring IGFAM. It is advantageous to create a nucleotide sequence encoding IGFAM or a derivative thereof having substantially different codon usage, such as including non-naturally occurring codons. 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 IGFAM and its derivatives without altering the encoded amino acid sequence is that it has a longer half-life than transcripts generated from naturally occurring sequences. This is to create an RNA transcript having the desired properties.

The present invention also includes the production of DNA sequences encoding IGFAM and derivatives of IGFAM, 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 IGFAM or any fragment thereof.

Also included in the invention are hybrids, under various stringent conditions, to the claimed polynucleotide sequences, and particularly to SEQ ID NOs: 20-38 and fragments thereof. Soyable polynucleotide sequences are included (see, eg, Wahl, G.
M. and SL Berger (1987) Methods Enzymol. 152: 399-407; Kimmel, AR (1987).
Methods Enzymol. 152: 507-511). The hybridization conditions include annealing and washing conditions described in the "Definition".

[0103] DNA sequencing methods are well known and can be used 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
Use enzymes such as a combination of proofreading exonuclease and 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 IGFAM can be extended using partial nucleotide sequences in a variety of PCR-based methods well known in the art to cleave upstream sequences such as promoters and regulatory elements. Can be detected. 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, Sarkar, G. (1993)). See 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, the capture PCR method includes PCR amplification of a DNA fragment adjacent to a known sequence of human and yeast artificial chromosomal DNA (for example, L
agerstrom, 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, walking within genomic DNA is possible using PCR, nested primers, and a PROMOTERFINDER library (Clonetech, Palo Alto, CA). This method eliminates the need to screen the library and is useful for finding intron / exon transitions. All PCs
The method R method based, primer, OLIGO ^TM 4.06 Primer Analy
Using commercially available software such as sis software (National Biosciences Inc., Plymouth, MN) or another suitable program, the GC is 22-30 nucleotides in length.
The content can be designed to anneal to the mold at a temperature of about 50% or more and at a temperature of about 68C to 72C.

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 that often contains the sequence of the 5 ′ region of a gene is an oligo d (T)
This is suitable when a full-length cDNA cannot be obtained from the library. 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 IGFAM, or a fragment thereof, is used in a recombinant DNA molecule to express IGFAM, 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 IGFAM.

To modify the sequence encoding IGFAM for a variety of reasons, including but not limited to modifying the cloning, processing and / or expression of the gene product,
The nucleotide sequences of the present invention can be manipulated using methods well known to those skilled in the art. 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.

In another embodiment of the invention, the entire sequence encoding IGFAM, or a portion thereof, can be synthesized using chemical techniques well known to those skilled in the art (eg, Caruther
sMH et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223; Horn, T. et al. (1980) Nucl.
Res.Symp.Ser.225-232). Alternatively, IGFAM 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: 202-20).
4). 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 IGFAM 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 preparative high performance liquid chromatography (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 IGFAM, the nucleotide sequence encoding IGFAM or a derivative thereof is converted to a suitable expression vector (ie, necessary for the transcription and translation of the coding sequence inserted into a suitable host). Vector containing the element). These elements include enhancers, constitutive and inducible promoters in the vector and the polynucleotide sequence encoding IGFAM, 5 'and 3'
And regulatory sequences such as the untranslated region of The length and specificity of such elements can vary. Specific initiation signals can be used to effect more efficient translation of sequences encoding IGFAM. Such signals include the ATG initiation codon and adjacent sequences such as, for example, the Kozak sequence. When the sequence encoding IGFAM, its initiation codon, and upstream regulatory sequences are inserted into the appropriate expression vector, additional transcriptional and translational regulatory signals may be unnecessary. However, if only the coding sequence or a fragment thereof is inserted, exogenous translational control signals, including the ATG start codon in the reading frame, 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, eg, Scharf,
(See D. et al. (1994) Results Probl. Cell Differ. 20: 125-162).

Expression vectors containing IGFAM coding sequences and appropriate transcriptional and translational regulatory regions
In order to produce, methods well known to those skilled in the art are used. These methods include:in v itro Recombinant DNA technology, synthetic technology, andin vivoIncludes genetic recombination techniques (eg,
See, Sambrook, J. et al. (1989)Molecular Cloning, A Laboratory Manual, Cold Spr
ing Harbor Press, Plainview, NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biology , John Wiley & Sons, New York, NY,
 ch. 9, 13 and 16).

A variety of expression vector / host systems are available for containing and expressing sequences encoding IGFAM. 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 purpose for which the polynucleotide sequence encoding IGFAM will be used. For example, IGFAM
Routine cloning, subcloning, and propagation of a polynucleotide sequence encoding a PBLUESCRIPT (Stratagene, La Jolla CA) or pSportl plasmid
(Life Technologies) or other multifunctional E. coli vectors. Ligation of the sequence encoding IGFAM into the 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 provide for in vitro transcription, dideoxy sequencing,
Useful for single-stranded rescue by helper phage and generation of nested deletions (
For example, Van Heeke. G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-55.
09). For example, when a large amount of IGFAM is required for the purpose of antibody production or the like, a vector that provides high-level IGFAM expression can be used. For example, a strong triggering T
Vectors containing the 5 or T7 bacteriophage promoter can be used.

A yeast expression system can be used to produce IGFAM. Yeast Saccharomyces cerevisiae
Alternatively, in Pichia pastoris , various vectors containing a constitutive or inducible promoter such as α-factor, alcohol oxidase, and PGH can be used. In addition, such vectors direct either 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 Enzymo
l. 153: 516-54; see Scorer, CA et al. (1994) Bio / Technology 12: 181-184).

[0116] Plant systems can also be used for the expression of IGFAM. Transcription of the sequence encoding IGFAM 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)).
EMBO 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.191-1
96).

[0117] In mammalian cells, a number of viral-based expression systems are available. If an adenovirus is used as an expression vector, the sequence encoding IGFAM may 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 IGFAM in host cells (eg, Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. . 81:36
55-3659). 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 provision 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 mammalian recombinant proteins, stable expression of IGFAM in a cell line is preferred. For example, using an expression vector
It is possible to transform a sequence encoding GFAM into a cell line, the expression vector comprising a replication and / or endogenous expression element 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.

[0120] 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, if a sequence encoding IGFAM is inserted within a marker gene sequence, transformed cells containing the sequence encoding IGFAM can be identified by the absence of marker gene function. Alternatively, a sequence encoding IGFAM and a 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, various methods well known to those skilled in the art can be used to identify host cells that contain a nucleic acid sequence encoding IGFAM and express IGFAM. 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 IGFAM 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 utilizing monoclonal antibodies reactive to two non-interfering epitopes on IGFAM
ay) 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); Sero logical Methods, a Laboratory Manual , APS Press, St Paul, MN Section IV; C
oligan, JE et al. (1997) Current Protocols in Immunology , Greene Pub.
iates and Wiley-lnterscience, New York, NY; and Pound, JD (1998) Immuno chemical 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 and amino acid assays. For detecting a sequence related to a polynucleotide encoding IGFAM, means for producing a labeled hybridization probe or PCR probe include oligolabeling (oligolabeling),
This includes nick translation, end-labeling, or PCR amplification using labeled nucleotides. Alternatively, the sequence encoding IGFAM, 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, for example,
RNA probes can be synthesized in vitro by adding a suitable RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. These methods are described in various commercially available kits (Amersham Pharmacia Biotech, Promega (Madison W
I), 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.

[0125] Host cells transformed with a nucleotide sequence encoding IGFAM 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 of skill in the art, expression vectors containing a polynucleotide encoding IGFAM can be designed to include a signal sequence that directs IGFAM 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 invention, a natural nucleic acid encoding IGFAM, a modified nucleic acid,
Alternatively, the recombinant nucleic acid sequence can be linked to a heterologous sequence which translates into the fusion protein of any of the aforementioned host systems. For example, a chimeric IGFAM protein containing a heterologous moiety that can be identified by a commercially available antibody can facilitate screening of a peptide library for inhibitors of IGFAM 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, hemagglutinin (HA)
Is included. GST, MBP, Trx, CBP, and 6-His allow purification of homologous fusion proteins with each of immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal chelating resins. 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 IGFAM and the heterologous protein sequence, and after purification, IGFAM can be cleaved from the heterologous portion. 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 IGFAM can be synthesized in vitro using a TNT rabbit reticulocyte lysate or 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,
Fragments of IGFAM 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). The various fragments of IGFAM can also be synthesized separately and then combined to create the full length molecule.

There are chemical and structural similarities, for example, in the context of sequences and motifs between therapeutic human immunoglobulin superfamily proteins and regions of IGFAM. In addition, IGFAM expression has implications for proliferative and cancerous tissues and hematopoiesis, inflammation, and other processes mediated by the immune system. Therefore, IGFAM
Is thought to play a role in cancer, immune system abnormalities and infectious diseases. In treating a disease associated with an increase in the expression or activity of IGFAM, it is desirable to reduce the expression or activity of IGFAM. In treating a disease associated with a decrease in the expression or activity of IGFAM, it is desirable to increase the expression or activity of IGFAM.

Thus, in one embodiment, IGFAM or a fragment or derivative thereof can be administered to a patient for the treatment or prevention of a disease associated with reduced IGFAM expression or activity. Examples of such diseases include, but are not limited to, adenocarcinoma, melanoma,
Sarcomas and teratocarcinomas, specifically, adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder,
Ganglia, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,
Cancers of the prostate, salivary glands, skin, spleen, testis, thymus and uterus; and inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS) and adrenal insufficiency, adult respiratory distress syndrome, allergy, ankylosing spondylitis , Amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease , Atopic dermatitis, dermatomyositis,
Diabetes, emphysema, erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, Irritable bowel syndrome, lymphocotoxin transient lymphopenia, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
Myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis, psoriasis,
Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenia, ulcerative colitis, Werner syndrome, cancer, hemodialysis And immune system abnormalities such as trauma, hematopoietic cancer including lymphoma, leukemia and myeloma; and adenovirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus, hepa Donavirus, herpes virus,
Flavivirus, orthomyxovirus, parvovirus, papovavirus,
Infection by viral pathogens classified as paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus and togavirus, and pneumococci, staphylococci, streptococci, bacilli, corynebacterium, clostridium , Neisseria meningitidis, Neisseria gonorrhoeae, Listeria, Moraxella, Kingella, Haemophilus, Legionella, Bordetella, Gram-negative enterobacteria (including Shigella and Salmonella, Campylobacter), Pseudomonas, Vibrio, Brucella, Francisella, Yersinia, Bartonella, norcardium, Actinomyces, Mycobacteria, Spirohetares, Rickettsia, Chlamydia and infection by bacterial pathogens classified as Mycoplasma, Aspergillus, Blastmyces, Dermatophytes, Cryptococcus, Infection by fungal pathogens classified as coccidioides, malasezzia, histoplasma and other fungal pathogens causing mycosis, and endophytic amoebae causing plasmodium or malaria, leishmania, trypanosoma, toxoplasma, pneumocystis carinii, giardia Intestinal protozoa such as genus, histological nematodes such as Trichomonas and Trichinella, intestinal nematodes such as roundworm, lymphatic filarial nematodes, and linear animals such as schistosomes and tapeworms. Includes infectious diseases such as infections by parasites.

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

In another embodiment, purified IGFAM together with a suitable pharmaceutical carrier is used to treat or prevent a disease associated with reduced expression or activity of IGFAM, including but not limited to those described above. Pharmaceutical compositions comprising the same may be administered to a patient.

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

In yet another embodiment, an antagonist of IGFAM may be administered to a patient for the treatment or prevention of a disease associated with increased IGFAM expression or activity. Examples of such diseases include, but are not limited to, the cancers, immune system abnormalities and infectious diseases described above. In one embodiment, an antibody that specifically binds IGFAM 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 IGFAM.

In another example, the complementary sequence of a polynucleotide encoding IGFAM can be expressed for the treatment or prevention of a disease associated with increased expression or activity of IGFAM, 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. IGFAM antagonists can be prepared using methods well known to those skilled in the art. In particular, a library of drugs can be screened using purified IGFAM to generate antibodies or to identify those that specifically bind to IGFAM. IGFAM antibodies 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 produce antibodies, various hosts, including goats, rabbits, rats, mice, humans, and the like, are immunized by injecting IGFAM or any fragment thereof having immunological properties or oligopeptides thereof. Can be 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 Cor ynebacterium parvum are especially preferable.

The oligopeptides, peptides or fragments thereof used to elicit antibodies for IGFAM have 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 a portion of the amino acid sequence of the native protein and include the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of IGFAM amino acids can be fused with stretches of another protein, such as KLH, to produce chimeric molecule antibodies.

[0140] Monoclonal antibodies to IGFAM can be prepared using any technique that produces 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: 495-49).
7; Kozbor, D. et al. (1985) Immunol. Methods 81: 31-42; Cote, RJ et al. (1983) P
roc. Natl. Acad. Sci. 80: 2026-2030; Cole, SP et al. (1984) Mol. Cell Biol.
62: 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 IGFAM. Antibodies with related specificities but differing 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).

[0142] 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. Na
86: 3833-3837; Winter, G. et al. (1991) Nature 349: 293-299).
.

[0143] Antibody fragments which contain specific binding sites for IGFAM 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, Fab expression libraries 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).

Various 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 IGFAM and its specific antibody. A two site monoclonal antibody-based immunoassay using a monoclonal antibody reactive to two non-interfering IGFAM epitopes.
Ased immunoassay) is usually used, but competitive binding assays can also be used (Pound, supra ).

Using various methods such as Scatchard analysis with radioimmunoassay techniques, IGF
Evaluate the affinity of the AM 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 IGFAM epitopes represents the average affinity or avidity of the IGFAM antibody. The Ka measured for a monoclonal antibody reagent monospecific for a particular IGFAM 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 for immunoassays in which the IGFAM antibody complex must withstand rigorous manipulations. Low-affinity antibody reagents with Ka values of 10 ⁶ to 10 ⁷ L / mol can be used for immunopurification (
It is preferably used for immunopurification and similar treatments. (Catty, D. (1988
) Antibodies, Volume I: A Practical Approach .IRL Press, Washington, DC;
Liddell, JE and Cryer, A. (1991) A Practical Guide to Monocional Anti bodies , John 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, polyclonal antibody reagents containing at least 1-2 mg / ml of specific antibody, preferably 5-10 mg / ml of specific antibody, are commonly used in methods requiring precipitation of the IGFAM antibody complex. . Guidelines for antibody specificity, titer, and avidity in various applications, as well as guidelines for antibody quality and use, are generally available
(See e.g., Catty, supra, and Coligan et al., Supra).

In another embodiment of the invention, a polynucleotide encoding IGFAM, or any fragment or complement thereof, can be used for therapeutic purposes. In one aspect, the mR
In situations where it is desirable to inhibit transcription of NA, a complementary sequence to a polynucleotide encoding IGFAM can be used. In particular, cells can be transformed with a sequence complementary to a polynucleotide encoding IGFAM. Accordingly, complementary molecules or fragments can be used to regulate IGFAM activity 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 IGFAM.

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 IGFAM can be produced using methods well known to those skilled in the art (see, eg, Sambrook, supra , and Ausubel, 1995, supra) . ).

A gene encoding IGFAM can be created by transforming a cell or tissue with an expression vector that expresses a polynucleotide or fragment thereof encoding IGFAM 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 regulatory, 5 ', or regulatory regions of the gene encoding IGFAM, 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 IGFAM.

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 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site will have secondary structural features that render the oligonucleotide inoperable. Can be evaluated. The suitability of the candidate target is determined by a ribonuclease protection assay (ribonase
uclease protection assay) and can be evaluated by inspection of the accessibility to hybridization with complementary oligonucleotides.

[0153] Complementary ribonucleic acid molecules and ribozymes of the invention can be produced 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, RNA molecules can be created by in vitro and in vivo transcription of a DNA sequence encoding IGFAM. 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.

[0154] 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, queosine, and wybutosine.

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 include IGFAM, IGFAM
Antibodies, mimetics, agonists, antagonists or inhibitors of IGFAM. 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.

[0158] The administration route of the pharmaceutical composition used in the present invention is not limited to the following, but may be oral administration, intravenous administration, intramuscular administration, intraarterial administration, intramedullary administration, intrathecal administration,
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 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 (optionally after grinding), treating the resulting mixture of granules in tablets or dragee cores (drafts).
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).

Formulations 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 conventional 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 compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. In the case of administration of IGFAM, such labels indicate the dosage, frequency of administration, and method of administration.

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 includes, for example, IGFAM or a fragment thereof that ameliorates a symptom or condition.
, An antibody of IGFAM, an agonist of IGFAM, an antagonist of IGFAM, or an antibody of IGFAM.
Refers to the amount of the active ingredient of the inhibitor. Therapeutic efficacy and toxicity can be determined by cell culture or
Standard pharmaceutical methods in animals, such as ED₅₀(Therapeutic in 50% of the population
Effective dose) or LD₅₀(50% lethal dose in the population)
Can be determined. The dose ratio of toxic to therapeutic effects is the therapeutic index and the LD ₅₀ / ED₅₀It can be expressed as a ratio. Pharmaceutical compositions exhibit a large therapeutic index.
Is preferred. Data obtained from cell culture assays and animal studies were
It can be used to summarize the range of dosage for use. Such ingredients
The dose of ED is low or no toxicity₅₀Including circulating concentrations of
It is preferably within the range. Depending on the dosage form used, patient sensitivity and route of administration
Thus, dosages will vary within this range.

[0171] 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, depending on 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 IGFAM may be used to diagnose a disease characterized by the expression of IGFAM or to treat a patient being treated with IGFAM or an agonist, antagonist or inhibitor of IGFAM. 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. Assays for the diagnosis of IGFAM include methods using antibodies and labels to detect IGFAM in extracts of human body fluids, cells or tissues. 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 are known in the art for measuring IGFAM, including ELISA, RIA and FACS, and provide the basis for diagnosing alterations or abnormal levels of IGFAM expression. . A normal, or standard, value for expression of IGFAM is that the antibody of IGFAM binds to a body fluid or cell extract obtained from a normal patient of a mammal (eg, a human subject) 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 IGFAM expressed in control and diseased samples from the patient's biopsy tissue was
Compare with standard value. Establish parameters for disease diagnosis by deviation between standard values and patient values.

In another embodiment of the invention, a polynucleotide encoding IGFAM can be used for diagnostic purposes. Polynucleotides used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. This polynucleotide is used to detect and quantify gene expression in biopsy tissues where IGFAM expression would be associated with disease. Diagnostic assays can be used to identify whether IGFAM is present, absent, or overexpressed, and to monitor modulation of IGFAM levels during therapeutic treatment.

In one aspect, nucleic acid sequences encoding IGFAM can be identified using hybridization with a PCR probe capable of detecting a polynucleotide sequence, including a genomic sequence, encoding IGFAM or a closely related molecule. . 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 region of interest or a less specific region (eg, a conserved motif). Depending on the nature, the probe
It is determined whether to identify only the naturally occurring sequence encoding M, or to identify 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 IGFAM. The hybridization probe of the present invention is DNA or RNA, or derived from the sequence of SEQ ID NO: 20-38, or an intron of the IGFAM gene,
It may be derived from a genome sequence including an enhancer and a promoter.

Means for generating specific hybridization probes for DNA encoding IGFAM include cloning a polynucleotide sequence encoding IGFAM or an IGFAM derivative into a vector to create an mRNA probe. is there.
Such vectors are well known to those of skill in the art and are commercially available, and can be used to generate RNA in vitro by adding an appropriate RNA polymerase or appropriate labeled nucleotides.
It can be used to synthesize a probe. 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 IGFAM can be used for diagnosis of a disease associated with expression of IGFAM. Examples of such diseases include, but are not limited to, adenocarcinoma, melanoma, sarcoma and teratocarcinoma, specifically, adrenal gland, bladder, bone, bone marrow,
Brain, breast, neck, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary,
Cancers of the pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus and uterus; and inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS) and adrenal insufficiency, adult respiratory distress syndrome , Allergy, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis , Contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, erythroblastosis, erythema nodosum, atrophic gastritis,
Glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome, lymphocotoxin temporary lymphopenia, mixed Type connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome , Rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenia, ulcerative colitis, Werner syndrome, cancer,
Immune system abnormalities such as complications of hemodialysis and extracorporeal circulation, trauma, hematopoietic cancers including lymphoma, leukemia and myeloma; and adenovirus, arenavirus, bunyavirus, calichevirus, coronavirus, filovirus By viral pathogens classified as hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus and togavirus Infections, pneumococci, staphylococci, streptococci, bacilli,
Corynebacterium, Clostridium, Meningococcus, Neisseria gonorrhoeae, Listeria, Moraxella, Kingella, Haemophilus, Legionella, Bordetella, Gram-negative enterobacteria (including Shigella and Salmonella, Campylobacter), Pseudomonas, Vibrio, Brucella, Francisera, Yersinia, Bartonella, norcardium
, Actinomyces, Mycobacteria, Spiroheteres, Rickettsia, Chlamydia, Mycoplasma, and other bacterial pathogens, Aspergillus, Blastmyces, Dermatophytes, Cryptococcus, Coccidioides, malasezzia
, Infection by fungal pathogens classified as histoplasma and other fungal pathogens that cause mycosis, and endophytic amoebae that cause plasmodium or malaria, Leishmania, Trypanosoma, Toxoplasma, Pneumocystis-carini, Giardia, etc. Parasites classified into tissue protozoa such as intestinal protozoa, Trichomonas and Trichinella, intestinal protozoa such as roundworm, lymphatic filarial nematodes, and nematodes such as schistosomes and tapeworms. Infectious diseases such as infections are included. Polynucleotide sequences encoding IGFAM can be analyzed by Southern blot or Northern analysis using patient biopsy tissue or body fluids, dot blot or other membrane-based techniques, PCR techniques, dipstick assays (di
pstick), pin and multi-format ELISA-like assays and microarrays can be used to detect changes in IGFAM expression. Such qualitative or quantitative test methods are well known to those skilled in the art.

In certain embodiments, nucleotide sequences encoding IGFAM may be useful, particularly in assays that detect the presence of related disorders as described above. The nucleotide sequence encoding IGFAM 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 IGFAM 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 related to the expression of IGFAM,
That is, a standard expression profile is established. This is accomplished by combining a body fluid or cell extract from a normal patient, either an animal or a human, with a sequence encoding IGFAM or a fragment thereof under conditions suitable for hybridization or amplification. Standard hybridization can be quantified by comparing values obtained from a normal patient to values obtained from experiments using known amounts of substantially purified polynucleotides. 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.

[0182] 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.

For cancer, the presence of abnormal amounts of transcripts (insufficiently 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 .

A further diagnostic use of an oligonucleotide designed from a sequence encoding IGFAM involves the use of PCR. These oligomers may be chemically synthesized, made with enzymes, or made in vitro . The oligomer is preferably a fragment of a polynucleotide encoding IGFAM, or IGFAM.
And a fragment of a polynucleotide complementary to the polynucleotide encoding is used under optimized conditions to identify a particular gene or condition. 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 IGFAM expression also include the use of radiolabeled or biotinylated nucleotides, the use of co-amplification of control nucleic acids,
And use of experimental results interpolated with standard calibration curves (eg, Melby, PC
(1993) J. Immunol. Methods, 159: 235-244; Duplaa, C. et al. (1993) Anal. Bio
chem. 229-236). The rate of quantification of large numbers of samples is such that the oligomer of interest is present in various dilute solutions and is quickly quantified by spectrophotometric or colorimetric response.
It can be accelerated by performing an ISA format 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.

[0187] Microarrays may be prepared and analyzed using methods well known to those skilled 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 IGFAM can be used to generate 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 product, wherein the artificial chromosome product comprises:
For example, there are human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructs or single-stranded chromosomal cDNA libraries (eg,
Harrington. JJ 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 position of the sequence encoding IGFAM on the physical chromosomal map and the specific disease,
Alternatively, association 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, the use of IGFAM, its catalytic or immunogenic fragments, or their oligopeptides, for screening libraries of compounds in various drug screening techniques. Can be. 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 IGFAM 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 IGFAM or a fragment thereof and washed. Next, the combined IGFAM is synthesized in a manner well known in the art.
Is detected. Also, purified IGFAM can be directly coated 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 a neutralizing antibody capable of binding IGFAM specifically competes with a test compound for binding IGFAM. In this way, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with IGFAM.

In yet another embodiment, the new technology is based on characteristics of currently known nucleotide sequences, including, but not limited to, the triplet genetic code and specific base pair interactions. If present, the nucleotide sequence encoding IGFAM,
It can be used for molecular biological techniques that have not yet been developed.

Those skilled in the art will be able to make full use of the present invention by the foregoing description without further explanation. Therefore, the specific preferred embodiments described below are merely illustrative and do not limit the invention.

All patent applications, patents, publications, and US patent applications [Attorney Specification No. PF-0643P] (filed on Nov. 19, 1998), US patent application Ser. No. 60 / 113,635 described herein.
And US Patent Application No. 60 / 128,194 are hereby incorporated by reference.

[0197]

【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), a single phase solution of guanidinium isothiocyanate and phenol. The resulting lysate was centrifuged on a CsCl cushion or extracted with chloroform. Ethanol with either isopropanol or sodium acetate and ethanol or in another conventional way from this lysate
RNA was precipitated.

[0198] RNA phenol extraction and precipitation were repeated as necessary to increase RNA purity. In some cases, RNA was treated with DNase. For most libraries, oligo
d (T) -bound paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatswort
hCA) 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 the POLY (A) PURE mRNA purification kit (Ambion, Austin TX).

In some cases, RNA was provided to Stratagene to create a corresponding cDNA library. Otherwise, UNIZAP ^™ vector system (Stratagene)
Alternatively, cDNA was synthesized using the SUPERSCRIPT ^™ plasmid system (Life Technologies) according to a recommended method known to those skilled in the art or a similar method, thereby preparing a cDNA library. (See, for example, Ausubel, 1997, supra , units 5.1-6.6). oligo d (
Reverse transcription was initiated using 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 SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech),
Or size of cDNA by agarose gel electrophoresis for separation (300-1000bp)
Was selected. Suitable plasmids (eg, pBLUESCRIPT (Stratagene), pSPORT1
Plasmid (Life Technologies) or pINCY (Incyte Pharmaceuticals Inc., Pa.
lo Alto CA), etc.) to the compatible restriction enzyme site of the polylinker. The recombinant plasmid is, for example, XL1-Blue, XL1-BlueMRF, or SOLR (Stratagen
e) or transformed into competent E. coli cells such as DH5α, DH10B, or ElectroMAX DH10B (Life Technologies).

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.

[0204] Validation of the polynucleotide sequence 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: 20-38. 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 technology 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 IGFAM 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 IGFAM The full-length nucleic acid sequence of SEQ ID NO: 20-38 was generated by extending an appropriate fragment of the full-length molecule and using oligonucleotide primers designed 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
Using 4.06 software (National Biosciences) or other suitable program, it is about 22-30 nucleotides in length, has a GC content of 50% or more, and has a temperature of about 68-72 ° C. The cDNA was designed to anneal to the target sequence. 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 extension is necessary or desirable, additional or nested sets of primers are 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.

The extended nucleotides were desalted and concentrated, transferred to a 384-well plate, and 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 are 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 NOs: 20-38, the above procedure, oligonucleotides designed for 5 ′ extension, and 5 ′ using appropriate genomic libraries
A regulatory sequence is obtained.

6 Labeling and Use of Individual Hybridization Probes Using the hybridization probes obtained from SEQ ID NOs: 20-38,
A, Screen 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.

The 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 Using microarray chemical coupling procedures and ink jet devices, array elements can be synthesized on the surface of the substrate. (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.

[0219] 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, may be obtained by a suitable method such as a glass slide. Place on the substrate. Its cDNA
Are fixed to glass slides, for example, by thermal and chemical treatment after UV crosslinking and further drying (eg, Schena, M. et al. (1995) Science 270: 467-
470; 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 IGFAM, or any portion thereof,
Used to detect and reduce, ie, suppress, GFAM expression. 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. OLIGO 4.06
Appropriate oligonucleotides are designed using software and the coding sequence of IGFAM. To repress transcription, complement oligonucleotides with the most unique 5 '
Designed from sequence and used to prevent binding of the promoter to the coding sequence. To suppress translation, complementary oligonucleotides are designed to prevent ribosome binding to the transcript encoding IGFAM.

9 Expression of IGFAM Expression and purification of IGFAM is performed using a bacterial or virus based expression system. For expression of IGFAM 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 the trp-lac (tac) hybrid promoter. The recombinant vector, BL
Transform into a suitable bacterial host such as 21 (DE3). Antibiotic resistant bacteria express IGFAM by induction with isopropyl β-D thiogalactopyranoside (IPTG).
Expression of IGFAM in eukaryotic cells is achieved by infecting insect or mammalian cell lines with Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The non-essential polyhedrin gene of the baculovirus is replaced with a cDNA encoding IGFAM 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 many cases, the recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells, but 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. US
A 91: 3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945).

In most expression systems, IGFAM is, for example, glutathione S-transferase (
GST), or a peptide epitope tag such as FLAG or 6-His, which is synthesized as a fusion protein, allowing 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, IGFAM to GST
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 IGFA obtained by these methods
M can be used directly in the following assays.

10 Demonstration of IGFAM activity In the assay for IGFAM activity, IGFAM was used to recognize and precipitate antigen from serum.
Measure your ability. This activity can be measured by a quantitative precipitation reaction (Go
lub, ES etal. (1987) Immunolony: A Synthesis , Sinauer Associates, Sun
derland, MA, pages 113-115). IGFAM is isotopically labeled by well-known methods. Various serum concentrations are added to a fixed amount of labeled IGFAM. The IGFAM-antigen complex is extracted as a precipitate in the solution and collected by centrifugation. The amount of precipitated IGFAM-antigen complex is proportional to the amount of radioisotope detected in the precipitate. Precipitating IGFAM-
The amount of antigen complex is plotted against serum concentration. Precipitin characteristic curves were obtained for various serum concentrations, where the amount of precipitating IGFAM-antigen complex initially increased in proportion to increasing serum concentration and peaked at the equivalence point with a maximum. From which it decreases with increasing serum concentrations. Thus, the amount of precipitating IGFAM-antigen complex is a measure of IGFAM activity characterized by sensitivity to both limiting and excess amounts of antigen.

Alternatively, assays for IGFAM activity measure the expression of IGFAM on the cell surface. The cDNA encoding IGFAM is transfected into a non-leukocyte cell line. Biotin labeling of cell surface proteins (de la Fuente. MA et al. (1997) Blood 90: 2398-24
05). Immunoprecipitation is performed using an antibody specific for IGFAM, and the immunoprecipitated sample is analyzed using SDS-PAGE and immunoblotting. Unlabeled immunoprecipitate
The ratio of labeled immunoprecipitant to (immunoprecipitant) is proportional to the amount of IGFAM expressed on the cell surface.

11 Functional Assays The function of IGFAM is to demonstrate that IGFAM functions at physiologically elevated levels in mammalian cell culture systems.
Assay by expression of the sequence encoding GFAM. 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 (Life Technologie) and pCR3.1 (Inv
itrogen, Carlsbad, CA), 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 (GFP;
Clontech), CD64, or CD64-GFP fusion protein. Automated laser optics based technology, flow cytometry (FCM), is used to identify transfected cells expressing GFP or CD64-GFP and to characterize the cells (eg,
Apoptotic status). 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, cell surface as measured by reactivity with specific antibodies And changes in the expression of protein in cells, as well as changes in plasma membrane composition as measured by binding of fluorescein-bound annexin V protein to the cell surface. For flow cytometry methods, see Ormerod, MG (1994) Flow Cytometry, Oxford, New
York, NY.

The effect of IGFAM on gene expression can be assessed using sequences encoding IGFAM as well as highly purified cell populations transfected with either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transformed cells and bind to conserved regions of human immunoglobulin G (IgG). The transformed cells are
Magnetic beads coated with either human IgG or CD64 antibodies can be used to separate from untransformed cells (DYNAL, Lake Success, NY). m
RNA can be purified from cells by methods well known to those skilled in the art. Expression of mRNA encoding IGFAM and other genes of interest can be analyzed by Northern analysis or microarray technology.

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

Alternatively, the amino acid sequence of IGFAM 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, oligopeptides 15 residues in length are 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 Naturally Generated IGFAM Using Specific Antibodies Naturally occurring or recombinant IGFAM is substantially purified by immunoaffinity chromatography using an antibody specific for IGFAM. The immunoaffinity column uses IGFAM antibody and CNBr-activated Sepharose (Amersham Pharmacia Biotech)
It is prepared by covalently binding to an activated chromatography resin as described above. After bonding, block and wash the resin according to the manufacturer's instructions.

The culture solution containing IGFAM is passed through an immunoaffinity column, and the column is washed under conditions that preferentially adsorb IGFAM (eg, a high ionic strength buffer in the presence of a surfactant). Conditions that disrupt antibody / IGFAM binding (eg, p
The column is eluted with a buffer of H2-3 or a high concentration of chaotrope, such as urea or thiocyanate ions, to recover IGFAM.

14 Identification of Molecules That Interact with IGFAM Label IGFAM or a biologically active fragment thereof with ¹²⁵ I Bolton-Hunter reagent (eg, Bolton AE and WM Hunter (1973) Biochem. 133: 52
9-539). Candidate molecules previously placed in the wells of a multiwell plate are incubated with labeled IGFAM, washed, and any wells with labeled IGFAM complexes are assayed. Using the data obtained at the various IGFAM concentrations, values for the binding, affinity and number of the IGFAM with the candidate molecule are calculated.

[0232] Those 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.

[0233] Briefly DESCRIPTION Table 1 Table, SEQ ID NO: (SEQ ID NO) of the polypeptide sequence and nucleotide sequence used to construct the full-length sequence encoding IGFAM, clone ID number (clone ID), 1 shows 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 IGFAM.

Table 3 shows selected fragments of each nucleic acid sequence, the tissue-specific expression pattern of each nucleic acid sequence determined by Northern analysis, the disease, disorder or condition associated with these tissues, and the DNA Indicates the cloned vector.

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

Table 5 shows the tools, programs and algorithms used for the analysis of IGFAM, along with appropriate descriptions, references and parameter thresholds.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[Table 12]

[Table 13]

[Table 14]

[Table 15]

[Table 16]

[Table 17]

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 1/16 A61P 3/10 4C085 1/18 5/14 4H045 3/10 7/00 5/14 7 / 06 7/00 9/00 7/06 9/10 101 9/00 11/00 9/10 101 11/06 11/00 13/12 11/06 17/00 13/12 17/06 17/00 17 / 12 17/06 19/02 17/12 19/06 19/02 19/10 19/06 21/04 19/10 25/28 21/04 29/00 25/28 101 29/00 31/00 101 31 / 04 31/00 31/10 31/04 31/12 31/10 31/18 31/12 35/00 31/18 35/02 35/00 37/00 35/02 37/02 37/00 37/08 37 / 02 43/00 111 37/08 C07K 16/18 43/00 111 16/42 C07K 16/18 C12N 1/15 16/42 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1 / 21 C12Q 1/68 A 5/10 G01N 33/53 M C12P 21/02 33/566 C12Q 1/68 C12N 15/00 ZNAC G01N 33/53 5/00 33/566 A61K 37/02 (31) Priority number 60/128, 194 (32) Priority date April 7, 1999 (1999.4.7) (33) Priority country United States (US) ( 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, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), 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, ZW (72) Inventor Korey, Neil Sea Mountain View, CA 40040, California, USA # 30 Dale Avenue 1240 (72) Inventor Gegler, Carl Jay 94025, California, United States of America U.S.A. Menlo Park Oakland Ave. 1048 (72) Inventor Gorgon, Gina A. 95006, California, U.S.A. (72) Inventor Bogun, Mariah Earl Sanrea, CA 94577, United States Doro Santiago Road 14244 (72) Inventor Ryu, Dung Aina M. 95136, California, United States of America 95 San Francisco Park, Belmont Place 55 (72) Inventor Lal, Pleity 95054, California, United States of America Santa Clara Las Drive 2382 (72) Inventor Hillman, Jennifer El 94040, California, Mountain View # 12, Monroe Drive 230 (72) Inventor Young, Junming 95129, California, United States San Jose Clarendon Street 7136 F-term (reference) 4B024 AA01 AA11 AA12 AA13 BA44 CA04 DA06 EA04 GA11 HA12 4B063 QA19 QQ46 QR08 QR36 QR42 QR56 QS25 QS34 QX07 4B064 AG26 AG27 CA10 CA19 CC24 DA13 DA14 DA15 4B065 AA26X AA57X AA72X AA90X AA93Y AB01 BA01 CA25 ZA04A02 ZA662 ZA682 ZA 752 ZA812 ZA892 ZA942 ZA962 ZA972 ZB052 ZB072 ZB112 ZB132 ZB152 ZB262 ZB272 ZB322 ZB332 ZB352 ZB382 ZC062 ZC352 4C085 AA13 AA14 BB31 CC02 CC04 CC05 CC22 DD22 EE01 4H045 AA10 AA72A

Claims (20)

[Claims] 1. A substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19 and fragments thereof. 2. A substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of claim 1. 3. An isolated and purified polynucleotide encoding the polypeptide of claim 1. 4. A mutant sequence of an isolated and purified polynucleotide having at least 90% or more polynucleotide sequence identity to the polynucleotide of claim 3. 5. An isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide of claim 3. 6. An isolated and purified polynucleotide having a sequence complementary to the polynucleotide of claim 3. 7. A method for detecting a polynucleotide, comprising: (a) hybridizing the polynucleotide of claim 6 with at least one nucleic acid of a sample to form a hybridization complex; (b) A method for detecting the hybridization complex, comprising a detection step in which the presence of the hybridization complex is correlated with the presence of the polynucleotide in the sample. . 8. The method of claim 7, further comprising amplifying the polynucleotide before said hybridization. 9. An isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 20-38 and fragments thereof. 10. An isolated and purified polynucleotide variant having at least 90% or more polynucleotide sequence identity to the polynucleotide of claim 9. 11. An isolated and purified polynucleotide having a sequence complementary to the polynucleotide of claim 9. 12. An expression vector comprising at least a fragment of the polynucleotide of claim 3. 13. A host cell comprising the expression vector according to claim 12. 14. A method for producing a polypeptide, comprising: (a) culturing the host cell according to claim 13 under conditions suitable for expression of the polypeptide; and (b) medium for the host cell. And recovering the polypeptide from the protein. 15. A pharmaceutical composition comprising the polypeptide of claim 1 together with a suitable pharmaceutical carrier. 16. A purified antibody that specifically binds to the polypeptide of claim 1. 17. A purified agonist of the polypeptide of claim 1. 18. A purified antagonist of the polypeptide of claim 1. 19. A method for treating or preventing a disease associated with a decrease in the expression or activity of IGFAM, which comprises administering an effective amount of the pharmaceutical composition of claim 15 to a patient in need of such treatment. A method comprising the step of administering. 20. A method for treating or preventing a disease associated with increased expression or activity of IGFAM, wherein an effective amount of the antagonist of claim 18 is administered to a patient in need of such treatment. A method that includes a process.