JP2011087587A - 21 human secreted proteins - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To detect, treat, and prevent medical diseases, disorders, and/or conditions by using secreted proteins or genes encoding them. <P>SOLUTION: The invention relates to novel human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. There are also provided vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating diseases, disorders, and/or conditions related to these novel human secreted proteins. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

(Field of Invention)
The present invention relates to newly identified polynucleotides and polypeptides encoded by these polynucleotides, antibodies that bind to these polypeptides,
The use of such polynucleotides, polypeptides, and antibodies, and their production.

(Background of the Invention)
Unlike bacteria, which exist as a single compartment surrounded by a membrane, human cells and other eukaryotic cells are subdivided into many functionally distinct compartments by the membrane. Each membrane-separated compartment, or organelle, contains different proteins that are essential for the function of the organelle. A cell uses a “selection signal”, an amino acid motif located within a protein, to target the protein to a specific cellular organelle.

One type of sorting signal called a signal sequence, signal peptide, or leader sequence directs a class of proteins to an organelle called the endoplasmic reticulum (ER). ER separates membrane-separated proteins from all other types of proteins. Once localized to the ER, both groups of proteins can be further directed to another organelle called the Golgi apparatus. Where Golgi
Proteins are distributed in vesicles, including secretory vesicles, cell membranes, lysosomes, and other organelles.

Proteins targeted to the ER by a signal sequence can be released into the extracellular space as secreted proteins. For example, vesicles containing secreted proteins can fuse with the cell membrane and release their contents into the extracellular space (a process called exocytosis). Exocytosis can occur constitutively or after receipt of an evoked signal. In the latter case, the protein is stored in secretory vesicles (or secretory granules) until exocytosis is induced. Similarly,
Proteins present on the cell membrane are also “linkers” that hold the protein to the membrane
Can be secreted into the extracellular space by proteolytic cleavage.

Despite significant progress in recent years, only a few genes encoding human secreted proteins have been identified. These secreted proteins include commercially valuable human insulin, interferon, factor VIII, human growth hormone, tissue plasminogen activator, and erythropoietin. Thus, given the broad role of secreted proteins in human physiology, there is a need to identify and characterize new human secreted proteins and the genes that encode them. This finding makes it possible to detect, treat and prevent medical diseases, disorders and / or conditions by using secreted proteins or genes encoding them.

(Summary of the Invention)
The present invention relates to novel polynucleotides and their encoded polypeptides. The invention further relates to vectors, host cells, antibodies, and recombinant and synthetic methods for producing the polypeptides and polynucleotides.
Also provided are diagnostic methods for detecting diseases, disorders and / or conditions associated with the polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders and / or conditions. The invention further relates to a screening method for identifying the binding partner of this polypeptide.
For example, the present invention provides the following items.
(Item 1) An isolated nucleic acid molecule comprising:
(A) a polynucleotide fragment of SEQ ID NO: X, or high to SEQ ID NO: X
Polynucleotide of the cDNA sequence contained in ATCC deposit number Z that can be hybridized
Defragmentation;
(B) A polypeptide fragment of SEQ ID NO: Y or a hybrid to SEQ ID NO: X
Encoded by the cDNA sequence contained in ATCC deposit number Z
A polynucleotide encoding a polypeptide fragment;
(C) the polypeptide domain of SEQ ID NO: Y, or hybridize to SEQ ID NO: X
A poly- sequence encoded by the cDNA sequence contained in ATCC deposit number Z
A polynucleotide encoding a peptide domain;
(D) a polypeptide epitope of SEQ ID NO: Y or a hybrida to SEQ ID NO: X
A sequence encoded by the cDNA sequence contained in ATCC deposit number Z
A polynucleotide encoding a repeptide epitope;
(E) a polynucleotide encoding the polypeptide of SEQ ID NO: Y having biological activity
Included in a nucleotide or ATCC deposit number Z that can hybridize to SEQ ID NO: X
CDNA sequence to be wound;
(F) a polynucleotide which is a variant of SEQ ID NO: X;
(G) a polynucleotide which is an allelic variant of SEQ ID NO: X;
(H) a polynucleotide encoding a species homologue of SEQ ID NO: Y;
(I) any one of the polynucleotides specified in (a) to (h)
A polynucleotide capable of hybridizing under stringent conditions, comprising
Here, the polynucleotide is a nucleotide sequence having only A residues or only T residues.
Polynucleotides that do not hybridize to stringent nucleic acid molecules under stringent conditions
Creotide,
Having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of
An isolated nucleic acid molecule comprising a polynucleotide.
(Item 2) The polynucleotide fragment binds a secreted protein.
2. An isolated nucleic acid molecule according to item 1, comprising a nucleotide sequence to be loaded.
(Item 3) The polynucleotide fragment is the same as SEQ ID NO: Y.
To the defined sequence or ATCC deposit number Z which can hybridize to SEQ ID NO: X
Nucleo encoding a polypeptide encoded by the contained cDNA sequence
2. The isolated nucleic acid molecule of item 1, comprising a tide sequence.
(Item 4) The polynucleotide fragment is the whole of SEQ ID NO: X.
Nucleotide sequence or ATCC deposit number that can hybridize to SEQ ID NO: X
2. The isolated nucleic acid molecule of item 1, comprising a cDNA sequence contained in Z.
(Item 5) Whether the nucleotide sequence is either C-terminal or N-terminal
3. The isolated nucleic acid molecule of item 2, comprising these consecutive nucleotide deletions.
(Item 6) The nucleotide sequence is from either the C-terminus or the N-terminus.
4. The isolated nucleic acid molecule of item 3, comprising the consecutive nucleotide deletions of
(Item 7) A recombinant vector comprising the isolated nucleic acid molecule according to item 1.
-.
(Item 8) A method for producing a recombinant host cell comprising the isolated nucleic acid molecule according to item 1.
(Item 9) A recombinant host cell produced by the method according to item 8.
(Item 10) The recombinant host cell according to item 9, comprising a vector sequence.
(Item 11) An isolated polypeptide comprising:
(A) of the encoded sequence contained in SEQ ID NO: Y or ATCC Deposit Number Z
Polypeptide fragments;
(B) Included in SEQ ID NO: Y or ATCC Deposit No. Z having biological activity
A polypeptide fragment of the encoded sequence;
(C) of the encoded sequence contained in SEQ ID NO: Y or ATCC Deposit Number Z
A polypeptide domain;
(D) of the encoded sequence contained in SEQ ID NO: Y or ATCC Deposit Number Z
A polypeptide epitope;
(E) of the encoded sequence contained in SEQ ID NO: Y or ATCC Deposit Number Z
Secretory form;
(F) of the encoded sequence contained in SEQ ID NO: Y or ATCC Deposit Number Z
Full length protein;
(G) a variant of SEQ ID NO: Y;
(H) an allelic variant of SEQ ID NO: Y; or
(I) a species homologue of SEQ ID NO: Y,
An amino acid sequence that is at least 95% identical to a sequence selected from the group consisting of:
Isolated polypeptide.
(Item 12) The secretory form or the full-length protein is C-terminal or
12. Isolation according to item 11, comprising consecutive amino acid deletions from any of the N-termini
Polypeptide.
(Item 13) Specifically bound to the isolated polypeptide according to item 11.
An isolated antibody that combines.
(Item 14) Expressing the isolated polypeptide according to item 11,
Recombinant host cell.
(Item 15) A method for producing an isolated polypeptide, comprising:
(A) The recombination according to item 14, under conditions such that the polypeptide is expressed
Culturing the host cell; and
(B) recovering the polypeptide,
Including the method.
(Item 16) A polypeptide produced by the method according to item 15.
(Item 17) A method for preventing, treating, or alleviating a medical condition, comprising the step of administering a therapeutically effective amount of the polypeptide of item 11 or the polynucleotide of item 1 to a mammalian subject. The method of inclusion.
(Item 18) A pathological condition or a pathological condition in a subject
A method for diagnosing susceptibility,
(A) presence or absence of mutation in the polynucleotide according to item 1
Determining; and
(B) a pathological condition or pathological condition based on the presence or absence of the mutation
Diagnosing susceptibility to the condition,
Including the method.
(Item 19) A pathological condition or a pathological condition in a subject
A method for diagnosing susceptibility,
(A) Presence of expression of the polypeptide of item 11 in a biological sample
Determining the presence or quantity, and
(B) a pathological condition based on the presence or amount of expression of the polypeptide, or
Diagnosing susceptibility to a pathological condition,
Including the method.
(Item 20) A binding partner for the polypeptide according to item 11
A method for identifying
(A) contacting the polypeptide of item 11 with a binding partner;
and
(B) determining whether the binding partner results in activity of the polypeptide
Process
Including the method.
(Item 21) A gene corresponding to the cDNA sequence of SEQ ID NO: Y.
22. A method for identifying activity in a biological assay comprising:
Where the method is as follows:
(A) expressing SEQ ID NO: X in the cell;
(B) isolating the supernatant;
(C) detecting activity in a biological assay; and
(D) identifying a protein in the supernatant having the activity;
Including the method.
(Item 23) A product produced by the method according to item 20.

(Detailed explanation)
(Definition)
The following definitions are provided to facilitate understanding of certain terms used throughout the specification.

In the present invention, “isolated” refers to a material that has been removed from its original environment (eg, the natural environment if it is naturally occurring), and thus its natural The state has been changed "by human hand". For example, an isolated polynucleotide can be part of a vector or composition of matter, or can be contained in a cell and still “isolated”. This is because the vector, composition of matter, or particular cell is not the natural environment for the polynucleotide. The term “isolated” refers to a genomic library or cDNA
Library, whole cell or mRNA preparation, genomic DNA preparation (
Including those separated by electrophoresis and transferred to a blot), sheared whole cell genomic DNA preparations, or other compositions for which the art does not demonstrate the distinguishing features of the polynucleotide / sequence of the present invention I do n’t need anything.

In the present invention, a “secreted” protein refers to a protein that can be directed as a result of a signal sequence into the ER, secretory vesicle, or extracellular space, as well as a protein that does not necessarily contain a signal sequence but is released into the extracellular space. . If a secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a “mature” protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.

In certain embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500 or at least 1000 contiguous nucleotides but are 300 kb, 200 kb, 100 kb in length 50 kb, 15 kb, 10 kb, 7.
It is 5 kb, 5 kb, 2.5 kb, 2.0 kb or 1 kb or less. In further embodiments, the polynucleotides of the invention, as disclosed herein, include a portion of a coding sequence, but do not include all or a portion of any intron. In another embodiment, the polynucleotide comprising the coding sequence does not include the coding sequence of the genomic flanking gene (ie, 5 ′ or 3 ′ to the gene of interest in the genome). In another embodiment, the polynucleotide of the invention is 10
00, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3,
It does not contain two or more than one genomic flanking gene coding sequence.

As used herein, “polynucleotide” refers to SEQ ID NO: X (SE
Q ID NO: X) refers to a molecule having a nucleic acid sequence included in the DNA, or a cDNA included in a clone deposited with the ATCC. For example, the polynucleotide may include 5 ′ and 3 ′ untranslated sequences, coding regions with or without signal sequences, nucleotide sequences of full-length cDNA sequences including secreted protein coding regions, and fragments, epitopes, domains of the nucleic acid sequences And variants. Further, as used herein, “polypeptide”, as broadly defined, refers to a molecule having a translated amino acid sequence derived from a polynucleotide.

In the present invention, the full-length sequence identified as SEQ ID NO: X was often generated by overlapping sequences contained in multiple clones (contig analysis).
A representative clone containing all or most of the sequence for SEQ ID NO: X has been deposited with the American Type Culture Collection (“ATCC”). As shown in Table 1, each clone is identified by cDNA clone ID (identifier) and ATCC accession number. ATCC has 10801 University
Boulevard, Manassas, Virginia 2011-10-22
09, located in USA. The ATCC deposit was made in accordance with the provisions of the Budapest Treaty concerning the international recognition of the deposit of microorganisms for patent procedure purposes.

The “polynucleotide” of the present invention also includes a polynucleotide capable of hybridizing under stringent hybridization conditions to the sequence contained in SEQ ID NO: X, its complement, or cDNA in a clone deposited with the ATCC. . “Stringent hybridization conditions” means 50% formamide, 5 × SSC (750 mM NaCl, 75 mM trisodium citrate), 5
Overnight incubation at 42 ° C. in a solution containing 0 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA, followed by 0.1 × SSC This is to wash the filter at about 65 ° C.

Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the invention at lower stringency hybridization conditions. Changes in hybridization stringency and signal detection are achieved primarily through manipulation of formamide concentration (lower percentages of formamide result in reduced stringency); salt conditions, or temperature. For example, lower stringency conditions are: 6 × SSPE (20 × SSPE = 3M NaCl;
2M NaH ₂ PO ₄ ; 0.02M EDTA, pH 7.4), 0.5% SDS
, Overnight incubation at 37 ° C. in a solution containing 30% formamide, 100 μg / ml salmon sperm blocking DNA; then 1 × SSPE, 0.1% S
Includes cleaning at 50 ° C. with DS. Furthermore, to achieve even lower stringency, washing performed after stringent hybridization can be performed at higher salt concentrations (eg, 5 × SSC).

Note that changes in the above conditions can be achieved by the inclusion and / or substitution of alternative blocking reagents used to suppress background in hybridization experiments. Exemplary blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available patent formulations. Inclusion of specific blocking reagents may require modification of the hybridization conditions described above due to compatibility issues.

Of course, a polynucleotide that hybridizes only to a poly A + sequence (eg, any 3 ′ terminal poly A + region (tract) of the cDNA shown in the sequence listing) or to a complementary stretch of T (or U) residues is It is not included in the definition of “polynucleotide”. Because such polynucleotides hybridize to any nucleic acid molecule comprising a poly (A) stretch or its complement (eg, virtually any double-stranded cDNA clone generated using oligo dT as a primer). It is because it soybeans.

The polynucleotide of the present invention may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or unmodified DN
A or modified RNA or modified DNA. For example, a polynucleotide is a single and double stranded DNA, a DNA that is a mixture of single and double stranded regions.
Single stranded and double stranded RNA, and RNA that is a mixture of single stranded and double stranded regions, single stranded, or more typically double stranded or a mixture of single stranded and double stranded regions. It can be composed of possible hybrid molecules including DNA and RNA. Furthermore, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also include one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA;
“Polynucleotide” includes chemically, enzymatically or metabolically modified forms.

The polypeptides of the present invention may be composed of amino acids linked to each other by peptide bonds or modified peptide bonds, ie, peptide isosteres, and may include amino acids other than the 20 amino acids encoded by the gene. This polypeptide is either by natural processes such as post-translational processing or by chemical modification techniques well known in the art.
Can be modified. Such modifications include basic texts and more detailed research papers,
As well as well documented in many research literature. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and the amino terminus or carboxyl terminus. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can contain many types of modifications. Polypeptides can be branched, for example, as a result of ubiquitination, and polypeptides can be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides can result from natural post-translational processes or can be made by synthetic methods. As a modification,
Acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond,
Covalent bond of heme moiety, covalent bond of nucleotide or nucleotide derivative, covalent bond of lipid or lipid derivative, phosphatidylinositol (phospho
idylinositol) covalent bond, crosslinking, cyclization, disulfide bond formation,
Demethylation, formation of covalent bridges, cysteine formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Pegylatio
n), transfer RNA-mediated addition of amino acids to proteins such as proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and ubiquitination. (For example, PROTEI
NS-STRUCTURE AND MOLECULAR PROPERITE
S, 2nd edition, T.E. E. Creighton, W.M. H. Freeman and
Company, New York (1993); POSTTRANSLATI
ONAL COVALENT MODIFICATION OF PROTEI
NS, B. C. Johnson, Academic Press, New Y
ork, pages 1-12 (1983); Seifter et al., Meth Enzymo.
l 182: 626-646 (1990); Rattan et al., Ann NY A
cad Sci 663: 48-62 (1992)).

“SEQ ID NO: X” refers to a polynucleotide sequence, whereas “SEQ ID NO: Y” refers to a polypeptide sequence. Identified by the integer specified in.

A “polypeptide having biological activity” refers to a polypeptide (including mature form) of the present invention, whether or not dose dependent, as measured in a specific biological assay. A polypeptide that exhibits activity that is similar to, but not necessarily identical to, activity. Where a dose dependency exists, it need not be identical to that of the polypeptide, but rather is substantially similar to the dose dependency in a given activity when compared to the polypeptide of the invention ( That is, the candidate polypeptide is
Exhibit greater activity or about 1/25 compared to a polypeptide of the invention
And preferably more than about 1/10 activity, and most preferably about 1/3.
It shows the above activity).

Many proteins (and translated DNA sequences) contain regions where the amino acid composition is highly offset with respect to a small subset of available residues. For example, the transmembrane domain and signal peptide (which is also transmembrane)
Includes long stretches dominated by leucine (L), valine (V), alanine (A), and isoleucine (I). Polyadenosine region at the end of cDNA (polyA
) Appears as polyphenylalanine (poly-F) when polylysine (poly-K) and reverse complement are translated in forward translation. These areas are often referred to as “lower complexity” areas.

Such a region may allow a database similarity search program such as BLAST to find high-scoring sequence matches that do not imply true homology. Most weight matrices (used to score alignments produced by BLAST) are generally of the group of hydrophobic amino acids (L, V and I) found in certain low complexity regions To match between either
The problem is exacerbated by the fact that a high score for an exact match gives a high score almost similar.

To correct for this, BLASTX. 2 (Version 2.0a5MP-
WashU “masks” lower complexity in a particular sequence.
) "Two filters (" seg "and" xnu ") are used. These filters analyze the sequence for such regions and create new sequences in which amino acids in lower complexity regions are replaced by the letter “X”. This is then the input sequence to the BLASTX program (sometimes referred to herein as “
Queries "and / or" Q "). This regimen helps to ensure that high score matches represent true homology, while the BLASTX program uses a query sequence masked by a filter to draw the alignment, resulting in negative results. Exists.

Thus, the stretch of “X” in the alignment shown in the following applications does not necessarily indicate that either the underlined DNA sequence or the translated protein sequence is unknown or unspecified. And also the presence of such a stretch is not meant to indicate that this sequence is identical or not identical to the sequence disclosed in the alignment of the invention. Such a stretch is defined by the BLASTX program as defined above.
Due to the detection of a low complexity region, it may simply indicate masking amino acids in that region. In all cases, the specification, the sequence listing (Table 1), and / or
Or the reference sequence shown in the deposited clone (sometimes referred to herein as “Subject”, “Sbjct”, and / or “S”) is a definitive implementation of the invention. Unless otherwise indicated in the specification and otherwise indicated elsewhere herein, the invention should not be construed to limit the invention to the partial sequences shown in the alignment.

(Polynucleotide and polypeptide of the present invention)
(Characteristics of protein encoded by gene number 1)
Using the computer algorithm BLASTX, the translation product of this gene is, as a non-limiting example, the accession number gb | AAD3389 in the following database:
2.1 | A sequence that is accessible through AF142780_1 (all information available through the accession numbers listed is hereby incorporated by reference) Mus
musculus] ”) and shared sequence homology. A partial alignment showing the observed homology is shown immediately below.

上記で「Ｓ」として示されるｇｂ｜ＡＡＤ３３８９２．１｜ＡＦ１４２７８０
＿１のセグメントは、配列番号として配列表中に示される。構造的な類似性に基
づいて、これらの相同なポリペプチドは、少なくともいくつかの生物学的活性を
共有することが予期される。このような活性は、当該分野で公知であり、そして
そのいくつかが本明細書中の他で記載される。このような活性を決定するための
アッセイもまた、当該分野で公知であり、そのいくつかが、本明細書中の他で記
載されている。本発明の好ましいポリペプチドは、上記で示されるアライメント
（コンピューターによって、配列に導入されたギャップは、もちろん除去される
）において、「Ｑ」配列に対応する配列番号として配列表に示されるアミノ酸配
列を有するポリペプチドを含む。

Gb | AAD33892.1 | AF142780, shown above as “S”
The segment of _1 is shown in the sequence listing as a sequence number. Based on structural similarity, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art and some are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein. A preferred polypeptide of the present invention comprises the amino acid sequence shown in the sequence listing as a SEQ ID NO corresponding to the “Q” sequence in the alignment shown above (gap introduced into the sequence by a computer is of course removed). Having a polypeptide.

This gene is mainly expressed in the following tissues / cDNA libraries:
Soares_pregnant_uterus_NbHPU, and to a lesser extent: Primary Dendritic Cells, l
ib 1; Thymus; Human Adult Spleen; Aorta
endothelial cells + TNF-a; Human T-c
ell lymphoma, re-exclusion; Primary Den
drictic cells, frac 2, and Soares_testis
_NHT.

The polynucleotides and polypeptides of the invention are for differential identification of tissues or cell types present in a biological sample, and the following: diseases and conditions including but not limited to diseases and / or disorders of the immune system It is useful as a reagent for diagnosis of Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For a number of disorders of the tissue or cells, particularly the immune system, significantly higher or higher than the standard gene expression level (ie, the level of expression in healthy tissue or body fluid from an individual without the disorder) A low level of expression of this gene indicates that certain tissues or cell types (eg, immune tissue, cancerous tissue and wound tissue) or body fluids (eg, lymph,
Serum, plasma, urine, synovial fluid and cerebrospinal fluid), or another tissue or sample,
It can be detected routinely.

Based on tissue distribution and homology to butyrophilin, polynucleotides, translation products and antibodies corresponding to this gene may be useful in the diagnosis and / or treatment of various disorders. Increased expression in the uterus is associated with the diagnosis of reproduction disorders and / or
Or suggests utility for studying treatment. The homology to butyrophilin suggests that the polynucleotides, translation products and antibodies corresponding to this gene may be useful in dairy products, vaccines and assays for susceptibility to multiple sclerosis. This tissue distribution also indicates enrichment in hematopoietic / immune tissues. Furthermore, this gene product shares homology to B7-H1, a novel member of the B7 family of T cell costimulatory molecules. This gene product may therefore be useful in the treatment and / or diagnosis of immune disorders.

(Characteristics of protein encoded by gene number 2)
The translation product corresponding to this gene shares sequence number homology with the murine Gremlin protein, which is thought to play a role in the inhibition of bone morphogenetic proteins (see, eg, Genbank accession number AAC40111). thing).

This gene is mainly expressed in the following tissues / cDNA libraries:
Osteoblasts, and to a lesser extent: Soares
_Senescent_fibroblasts_NbHSF; Jiabon
e marrow stroma; Human Osteoblasts II
; Human Thymus Stromial Cells; Soares m
elanocyte 2NbHM; HSA 172 Cells; Human
Osteoblastoma; Stroma cell TF274; Str
atagene fibroblast (# 937212); Human en
domestic strategic cells-treated with
progesterone; Human endometric strtro
mal cells-treated with stradiol; Hum
an adult (K. Okubo); Human endometric
stromal cells; NCI_CGAP_Gas4; Human Ga
ll Blader; Colon Tumor; Human 8 Week
Where Embryo; Colon Tumor II; Human Bo
ne Marlow Stromal Fibroblast; HUMAN S
CHWANOMA; Crohn's Disease; Pharynx Car
cinoma; Human Normal Cartilage Fracti
on IV; Human Pre-Differentiated Adipo
cytes; HSC172 cells; NCI_CGAP_Co12; hum
an corpus colisum; NCI_CGAP_Co9; Synov
ial hypoxia-RSF subtracted; Human Sto
mach, re-exction; Human Colon, re-exci
sion; NCI_CGAP_Alv1; Gessler Wilms tum
or; Monocyte activated, re-exclusion; 12
Week Old Early Stage Human, II; Human
Pancreas Tumor, Reexcision; Synovial
Fibroblasts (control); Bone Marrow Str
omal Cell, untated; NCI_CGAP_Co3; CHM
E Cell Line, updated; Colon Carcinom
a; NCI_CGAP_Co8; Human Placena; Pancre
as normal PCA4 No; Monocyte activated
; Pancreas Tumor PCA4 Tu; Neutrofils
IL-1 and LPS induced; Frontal corte
x, epilepic, re-exclusion, and NCI_CGAP_
Lu5.

The polynucleotides and polypeptides of the present invention may be used for differential identification of tissues or cell types present in a biological sample and for the following: diseases and including but not limited to musculoskeletal diseases and / or disorders Useful as a reagent for diagnosing conditions. Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For many disorders of the tissue or cells, particularly the musculoskeletal system, significantly higher than the standard gene expression level (ie, the level of expression in healthy tissue or fluid from an individual without the disorder), or Lower levels of expression of this gene indicate that certain tissues or cell types (eg, musculoskeletal tissue, cancerous tissue and wound tissue) or body fluids (eg, lymph, serum, In plasma, urine, synovial fluid and cerebrospinal fluid), or in another tissue or sample.

The homology of the translation product corresponding to this gene to the mouse gremlin protein allows the polynucleotide, translation product and antibody corresponding to this gene to be useful for the inhibition of members of the TGF-β family of growth factors. It shows that. Increased expression of this gene product in osteoblasts and osteoclast tissue indicates that polynucleotides, translation products and antibodies corresponding to this gene may play a role in osteoblast survival, proliferation and / or growth I suggest that. Thus, this gene product may be useful for effects on bone mass in conditions such as osteoporosis.

This secreted protein elicits antibodies to determine biological activity,
It can be used as a tissue marker, to isolate homologous ligands or receptors, to identify agents that modulate their interaction, and as a nutritional supplement. The protein can also have a very wide range of biological activities. Typical applications are the following “chemotaxis” and “binding activity” sections, Examples 11, 12, 1
3, 14, 15, 16, 18, 19 and 20 and elsewhere herein. Briefly, the protein may have the following activities: cytokines, cell growth / differentiation regulating activity, or induction of other cytokines; immunostimulatory / immunosuppressive activity (eg, human immunodeficiency virus infection, cancer Regulation of hematopoiesis (eg to treat anemia or as an adjunct to chemotherapy); stimulation or proliferation of bones, cartilage, tendons, ligaments, and / or nerves; (Eg, for treating wounds, for stimulation of follicle stimulating hormone (for control of fertility); chemotaxis and chemokinesis activity (eg,
Anti-inflammatory activity (eg to treat septic shock, Crohn's disease); antibacterial drugs As; to treat psoriasis or other hyperproliferative diseases; to regulate metabolism and behavior. The use of corresponding nucleic acids in gene therapy procedures is also contemplated.

The tissue distribution in immune cells (eg, neutrophils, bone marrow) indicates that the protein product of this clone is useful for diagnosis and treatment of various immune system disorders. Representative applications are described in Example 1 in the “Immunoactivity” and “Infectious Diseases” sections below.
1, 13, 14, 16, 18, 19, 20 and 27, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating hematopoietic cell line proliferation; survival; differentiation; and / or activation, including blood stem cells. Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests utility in the treatment of cancer (eg, by boosting the immune response). Expression in cells of lymphoid origin indicates that this natural gene product is involved in immune function. Therefore, it also has arthritis, asthma, AI
Immunodeficiency diseases such as DS, leukemia, rheumatoid arthritis, chronic granulomatosis, inflammatory bowel disease, sepsis, wound, neutropenia, neutropenia, psoriasis, hypersensitivity (e.g.
T cell mediated cytotoxicity); immune responses to transplanted organs and tissues (eg, host versus graft and graft versus host disease) or autoimmune disorders (eg, autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosus, It is also useful as a drug for immunological disorders, including drug-induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.

In addition, the protein may represent a secreted factor that affects the differentiation or behavior of other blood cells or that recruits hematopoietic cells to the site of injury. Thus, this gene product is thought to be useful in the expansion of various blood lineage stem cells and directed progenitor cells, and in the differentiation and / or proliferation of various cell types. Tissue distribution (eg, osteoblasts) suggests that the protein product of this clone is useful for the diagnosis and / or treatment of bone disorders. The tissue distribution in tumors of colon, pancreas and pharyngeal origins indicates that the protein product of this clone is
It suggests that it is useful for the diagnosis and inhibition of these tumors, as well as other tumors whose expression has been shown. In addition, this protein also raises antibodies to determine biological activity, in addition to its use as a nutritional supplement,
As a tissue marker, it can be used to identify agents that modulate their interaction to isolate homologous ligands or receptors. The protein and antibodies directed against this protein may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of protein encoded by gene number 3)
The translation product corresponding to this gene shares sequence homology with mouse MPS-1 protein and rat MPG-1 protein (see Genbank accession numbers AAA73957 and AAD38417, respectively). The MPS-1 protein is a macrophage-specific protein that has evolutionary conservation to the soluble family of perforin proteins. Based on homology, the translation product corresponding to this gene may function in lytic capacity and cause cell death.

This gene is mainly expressed in the following tissues / cDNA libraries:
Human Bone Marrow, treated, and to a lesser extent: Human rejected kidney;
T-cell lymphoma, re-exclusion; Human Ad
ultra Small Intestine; Human Osteoblast
s II; Ulcerative Colitis; Human Placen
ta; human tonsils; Hodgkin's Lymphoma
II, and Primary Dendritic Cells, lib 1.

The polynucleotides and polypeptides of the invention are for differential identification of tissues or cell types present in a biological sample, and the following: diseases and conditions including but not limited to diseases and / or disorders of the immune system It is useful as a reagent for diagnosis of Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For a number of disorders of the tissue or cells, particularly the immune system, significantly higher or higher than the standard gene expression level (ie, the level of expression in healthy tissue or body fluid from an individual without the disorder) A low level of expression of this gene indicates that certain tissues or cell types (eg, immune tissue, cancerous tissue and wound tissue) or body fluids (eg, lymph,
Serum, plasma, urine, synovial fluid and cerebrospinal fluid), or another tissue or sample,
It can be detected routinely.

This gene is a human ortholog of mouse and rat macrophage-specific gene 1. This mouse gene was identified by differential cDNA analysis and showed macrophage lineage and differentiation stage specific expression at high levels in mature macrophages and moderate levels in certain myelomonocytic cell lines.

Tissue distribution in human bone marrow and other hematopoietic cell types indicate that this gene and its protein products are useful for the diagnosis and treatment of human immune system disorders, osteoblastomas, arthritis and other cancers. The translation product corresponding to this gene can function as a lytic protein involved in cell death.

Alternatively, the translation product corresponding to this gene can participate in immune function and immune surveillance. Furthermore, the translation products corresponding to this gene, and antibodies against these translation products may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of protein encoded by gene number 4)
This gene is mainly expressed in the following tissues / cDNA libraries:
Soares fetal river screen 1NFLS and to a lesser extent: NCI_CGAP_GCB1; Human Pi
tuary, sub IX; Soares_NFL_T_GBC_S1;
Soares_NhHMPU_S1; Human Pituitary, sub
tracted; Soares adult brain N2b4HB55Y
Rejected Kidney, lib 4; Frontal cor
tex, epilepic, re-exclusion; Soares mel
anocyte 2NbHM; Human Cerebellum;
aated T-Cells, 12 hrs, subtracted; Human
Colon, subtraction; Pancreatic Islet;
NCI_CGAP_Ut4; Stratagene NT2 neuronal
precursor 937230; NCI_CGAP_Kid6; Stra
tagane pancreas (# 937208); Stratagene
HeLa cell s3 937216; Stratagene endot
helical cell 937223; NCI_CGAP_Co3; Stra
tagine colon (# 937204); Early Stage Hu
man Brain; Human Metal Kidney, Reexcis
ion; Human Fetal Heart; human tonsils;
NCI_CGAP_GC6; NCI_CGAP_Kid5; HUMAN B C
ELL LYMPHOMA; Soares overtime tutor NbHO
T; Hodgkin's Lymphoma II; Soares_testi
s_NHT; Leukocytes, normalized cot 50
A3; PRMIX; Namalwa Cells; HL-60, RA 4h, S
abstracted; NCI_CGAP_Pr8; NCI_CGAP_Pr9;
Human Pituitary, re-exclusion; Human Fe
tal Brain; stomach cancer (human); Huma
n Colon; Human retina cDNA Tsp509I-cl
eaves sub library; NCI_CGAP_Lip2; Human
Feat Splene; Smooth muscle, control,
re-exclusion; Epididymus, caput & co
rpus; cerebellum, Enzyme subtracted;
Human Lung; Epididymus, cauda; Human
Lung Cancer, re-exclusion; Healing gro
in wound-zero hr post-indication (con
trol); Human Epididymus; STROMAL-OSTE
OCLASTOMA; Synovial IL-1 / TNF stimulat
ed; Hepatocyte Tumor; pBMC stimula
ted w / poly I / C; Smooth muscle, IL1b i
nduced; Stratagene schizo brain S11; S
Ynoial hypoxia-RSF subtracted; NCI_C
GAP_Co14; B-cells (unstimulated); NLCAP
prostate cell line; Jurkat T-Cell, S
phase; Lymph node breast cancer; Hum
an Adult Small Intestine; Breast, Norm
al: (4005522B2); Stratagene long carci
noma 937218; Human Prostate; B-Cells; H
uman Brain, Striatum; Monocyte activat
ed, re-exclusion; Human Fetal Kidney; Hu
man Uterine Cancer; NCI_CGAP_Gas4; Hum
an Pancreas Tumor, Reexcision; Overy, C
anchor (9809C332): Poorly differentiated
d adenocarcinoma; Overy, Cancer: (40045
76 A8); Human Chondrosarcoma; Bone Mar
row Stromal Cell, untated; Human Thy
mus Stromal Cells; Soares breast 2NbH
Bst; Human Adrenal Grand Tumor; Human
Gall Blader; Smooth muscle, serum ind
uced, re-exc; Smooth muscle, serum trea
ted; breast lymph node CDNA library; H
human Placenta; H Macrophage (GM-CSF tr
eated), re-exclusion; Normal colon; NCI_
CGAP_GC4; Human Synchronous Sarcoma; Panc
reas normal PCA4 No; 12 Week Early St
age Human II, Reexcision; NCI_CGAP_Brn
23; Monocyte activated; Soares_placent
a_8to9weeks_2NbHP8to9W; Human Tests;
Bone Marlow Cell Line (RS4, 11); NCI_CG
AP_Lu5; Keratinocyte; Colon Normal III
Soares_fetal_heart_NbHH19W, and Prima
ry Dendritic Cells, lib 1.

The polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample, and the following: diseases and conditions, including but not limited to immune system diseases and / or disorders It is useful as a reagent for diagnosis of Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For a number of disorders of the tissue or cells, particularly the immune system, significantly higher or higher than the standard gene expression level (ie, the level of expression in healthy tissue or body fluid from an individual without the disorder) A low level of expression of this gene indicates that certain tissues or cell types (eg, immune tissue, cancerous tissue and wound tissue) or body fluids (eg, lymph,
Serum, plasma, urine, synovial fluid and cerebrospinal fluid), or another tissue or sample,
It can be detected routinely.

The observed tissue distribution suggests that this gene product may be useful in the diagnosis and / or treatment of various disorders. Expression in fetal liver and spleen suggests a potential role in hematopoiesis and in the survival, differentiation, and / or activation of blood cell lineages. Thus, this gene product may be involved in immune system dysfunction, susceptibility to infection, autoimmunity, leukemia / lymphoma, inflammation and immune regulation. Furthermore, the translation products corresponding to this gene, and antibodies against these translation products may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of protein encoded by gene number 5)
Using the computer algorithm BLASTX, the translation product of this gene is, as a non-limiting example, the accession number gb | AAC1721 of the following database:
7.1 | sequences accessible through all the information available through the listed accession numbers (herein incorporated by reference)
AF016603) guanosine-diphosphatase lik
e protein [denoted as Homo sapiens]) and was determined to share sequence homology. A partial alignment showing the observed homology is shown immediately below.

上記で「Ｓ」として示されるｇｂ｜ＡＡＣ１７２１７．１｜のセグメントは、
配列番号：として配列表中に示される。構造的な類似性に基づいて、これらの相
同なポリペプチドは、少なくともいくつかの生物学的活性を共有することが予期
される。このような活性は、当該分野で公知であり、そしてそのいくつかが本明
細書中の他で記載される。このような活性を決定するためのアッセイもまた、当
該分野で公知であり、そのいくつかが、本明細書中の他で記載されている。本発
明の好ましいポリペプチドは、上記で示されるアライメント（コンピューターに
よって、配列に導入されたギャップは、もちろん除去される）において、「Ｑ」
配列に対応する配列番号：として配列表に示されるアミノ酸配列を有するポリペ
プチドを含む。

The segment of gb | AAC17217.1 |, shown above as “S”, is
It is shown in the sequence listing as SEQ ID NO :. Based on structural similarity, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art and some are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein. Preferred polypeptides of the invention have a “Q” in the alignment shown above (gap introduced into the sequence by the computer is of course removed).
A polypeptide having the amino acid sequence shown in the sequence listing as SEQ ID NO: corresponding to the sequence is included.

This gene is mainly expressed in the following tissues / cDNA libraries:
Soares infant brain 1 NIB and to a lesser extent: Messenger cell, frac 1; Human
Umbilical Vein Endothelial cells, fra
c B, re-exction; Adipocytes, re-excisi
on; Fetal Heart, re-exclusion; Smooth mu
scle, IL1b induced; Human Osteosarcoma
Human ovarian cancer; NCI_CGAP_CLL1;
PC3 Prostate cell line; Soares melano
cyte 2NbHM and Primary Dendritic Cell
s, lib 1.

The polynucleotides and polypeptides of the present invention are for the differential identification of tissues or cell types present in biological samples and for the diagnosis of diseases and conditions including but not limited to immune system disorders: It is useful as a reagent.
Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For a number of disorders of the tissue or cells, particularly the immune system, significantly higher or higher than the standard gene expression level (ie, the level of expression in healthy tissue or body fluid from an individual without the disorder) Low levels of expression of this gene indicate that certain tissues or cell types (eg, cancerous tissue and wound tissue) or bodily fluids (eg, serum, plasma, urine, synovial fluid and the like) collected from individuals with such disorders Cerebrospinal fluid), or in another tissue or sample.

Based on homology to apyrase, diphosphatase and CD39 (which is thought to be involved in cell-cell interactions and aggregation), this protein may have a similar role in cell-cell interactions. Based on expression in the fetal brain, this gene and its products recognize, learn and other more advanced neural functions (eg memory)
It may be involved in the formation of the correct neural interaction required for This gene may therefore be useful in the diagnosis and treatment of such disorders as well as other neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, ALS and multiple sclerosis. In addition, the protein may be useful in the treatment of nerve injury, including spinal cord injury, where nerve regeneration is advantageous. Furthermore, the translation products corresponding to this gene, and antibodies against these translation products may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of protein encoded by gene number 6)
The translation product corresponding to this gene shares sequence homology with the human HER2 receptor protein (see, eg, Genbank accession number AAA75493). This gene is mainly expressed in the following tissues / cDNA libraries: NCI_CGAP_Pr28, and to a lesser extent: N
CI_CGAP_Pr23; Hodgkin's Lymphoma I; He
aling grain wound-zero hr post-inc
ision (control); Healing grind ound, 6
. 5 hours post indication; NCI_CGAP_Ut2;
NCI_CGAP_Pr2; NCI_CGAP_Kid6; Soares ad
ultra brain N2b5HB55Y; Hodgkin's Lympho
ma II; Soares_fetal_heart_NbHH19W, and Soares infant brain 1 NIB.

The polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample, and include but are not limited to: diseases and / or disorders of the immune system, and cancer It is useful as a reagent for diagnosis of diseases and conditions. Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. Is it significantly higher than the standard gene expression level (ie, the level of expression in healthy tissue or body fluid from an individual who does not have the disorder) for many disorders of the tissue or cells, particularly the immune system and cancerous tissue? Or a lower level of expression of this gene indicates that certain tissues or cell types (eg, immune tissue, cancerous tissue and wound tissue) or body fluids (eg, lymph, serum) collected from individuals with such disorders , Plasma, urine, synovial fluid and cerebrospinal fluid), or in another tissue or sample.

The homology to the human Her2 receptor and its tissue distribution in primary human prostate tumors and Hodgkin I lymphoma tissues indicate that this gene and its protein product are useful in the diagnosis and treatment of human prostate cancer, Hodgkin lymphoma, and other immune system disorders. Indicates usefulness.

(Characteristics of protein encoded by gene number 7)
The translation product corresponding to this gene shares sequence homology with the human Sterin-1 protein (see, eg, Genbank accession number AJ251973), and the extendin-like protein.

In certain embodiments, the polypeptides of the invention comprise or consist of the following amino acid sequence: MSDNAPASLESG
SSSTPTNCSTSSAIPQPGAATKPWRSKSLSVKHSAT
VSMLSVKPPGPAPRPRPPEAMKPAPNNNQKSMLEKLK
LFNSKGGSKAGEGEPGSRDSTSLERLELPFSFEEEEL
EAASRMLTTVGPPASSSPKIALKGIAQRTFSRRALTNK
KSSLKGNEKEKEQQREKDDKSKKDLAKRASVTERL
DLKEEPKEDPSGAAVPEMPKKSKIASFIPIPKGKLN
SAKKEPMAPHSSGIPKPGMKSMPGKSPSAPAPSKEG
ERSSRSGKLSSGLPQQKPQLDGRHSSSSSSLASSEGK
GPGGTTLNHSISSQTVSGSVTGTTQTTGSTNTVSVQLP
QPQQQYNHPNTATVAPFLYRSQTDTEGNTXTESSST
GVSVEPXHFPRLDSLLLWKNSLGKILRLGGCGQSNPH
ATMTQQGRRGREF (SEQ ID NO: 146). In addition, fragments and variants of these polypeptides (eg, the fragments described herein, at least 80%, 85%, 90%, 95%, 96%, 9% of these polypeptides)
Polypeptides that are 7%, 98% or 99% identical, and polypeptides encoded by polynucleotides that hybridize under stringent conditions to polynucleotides encoding these polypeptides, etc.) are also provided by the present invention. Is included. Antibodies that bind to the polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

This gene is mainly expressed in the following tissues / cDNA libraries:
Soares_pregnant_uterus_NbHPU, and to a lesser extent: 7TM-PNMIX; Human Osteoblas
ts II; Human Fetal Kidney, Reexcision and Soares melanocite 2NbHM.

The polynucleotides and polypeptides of the present invention may be used for differential identification of tissues or cell types present in a biological sample and for the following: diagnosis of diseases and conditions, including but not limited to reproductive diseases and / or disorders. Useful as a reagent for Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For many disorders of the tissue or cells, especially the reproductive system, significantly higher than or more than the standard gene expression level (ie, the level of expression in healthy tissue or body fluid from an individual without the disorder) A low level of expression of this gene indicates that certain tissues or cell types (eg, reproductive tissue, cancerous tissue and wound tissue) or body fluids (eg, lymph, serum, plasma, In urine, synovial fluid and cerebrospinal fluid), or in another tissue or sample.

Homology to the human Steerin-1 protein, and to extensin-like proteins, and homology to Zn finger transcription factors suggests that this gene plays a role in gene expression, regulation, and development. The tissue distribution in the human pregnancy uterus and the osteoblasts indicate that this gene and its protein product are useful for the diagnosis and treatment of female reproductive disorders and abnormal embryo development. Furthermore, the translation product corresponding to this gene,
In addition, antibodies against these translation products may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of protein encoded by gene number 8)
This gene is mainly expressed in pooled dendritic cells and Human Thymus Stoma Cells.

The polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample, and the following: diseases and conditions, including but not limited to immune system diseases and / or disorders It is useful as a reagent for diagnosis of Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For a number of disorders of the tissue or cells, particularly the immune system, significantly higher or higher than the standard gene expression level (ie, the level of expression in healthy tissue or body fluid from an individual without the disorder) A low level of expression of this gene indicates that certain tissues or cell types (eg, immune tissue, cancerous tissue and wound tissue) or body fluids (eg, lymph,
Serum, plasma, urine, synovial fluid and cerebrospinal fluid), or another tissue or sample,
It can be detected routinely.

The tissue distribution in human dendritic cells and thymic stromal cells indicates that this gene and its protein product are useful for diagnosis and treatment of human immune disorders. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes, which may suggest utility in the treatment of cancer (eg, by boosting the immune response). Since this gene is expressed in cells of lymphoid origin, this gene or protein, and antibodies to this protein, may show utility as tumor markers and / or immunotherapeutic targets for the tissues listed above. Thus, it is also used as a drug for immunological disorders including arthritis, asthma, immunodeficiency diseases (eg AIDS), leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, pressure ulcers, and psoriasis. obtain. In addition, the gene product may have commercial utility in the expansion of various blood lineage stem cells and directed precursors, as well as in the growth of various cell types. Furthermore, the translation products corresponding to this gene, and antibodies against these translation products may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of protein encoded by gene number 9)
The gene encoding the disclosed cDNA is believed to be present on chromosome 12. Therefore, the polynucleotide relating to the present invention is useful as a marker in linkage analysis for chromosome 12.

This gene is mainly expressed in the following tissues / cDNA libraries:
Soares_fetal_heart_NbHH19W, and to a lesser extent: Human Live, normal; Early St
age Human Brain; hypothalamus, frac
A; Activated T-cells; Human Tonsils, Li
b 2; human corpus colisum; Stratagene
lug carcinoma 937218; Stratagene fet
al spleen (# 937205); Human umbilical v
ein endothelial cells, IL-4 induced; M
acrophage (GM-CSF treated); Stratagene
live (# 937224); Bone marrow; Endothel
ial-induced; Human Amygdala; Stratagen
e lung (# 937210); Spleen, Chronic Lymph
otic leukemia; Human Bone Marrow, tr
eated; Soares_NhHMPu_S1; Primary Dender
itic Cells, lib 1, and Soares infant br
ain 1 NIB.

The polynucleotides and polypeptides of the present invention provide for the differential identification of tissues or cell types present in biological samples, and the following: diagnosis of diseases and conditions, including but not limited to developmental and immune system disorders Useful as a reagent for Similarly, polypeptides and antibodies to these polypeptides are
Useful for providing immunological probes for differential identification of tissues or cell types.
Is it significantly higher than the standard gene expression level (ie, the level of expression in healthy tissue or body fluid from an individual without the disorder) for many disorders of the tissue or cells, especially the immune and developmental systems? Or a lower level of expression of this gene indicates that certain tissues or cell types (eg, immune tissue, developmental tissue, cancerous tissue and wound tissue) or body fluids (eg, Lymph, serum, plasma, urine, synovial fluid and spinal fluid), or in another tissue or sample, can be routinely detected.

The tissue distribution in human fetal tissue shows that this gene and its protein product
It is useful for the diagnosis and treatment of developmental disorders and adult immune disorders and cancer. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes, which may suggest utility in the treatment of cancer (eg, by boosting the immune response). Since this gene is expressed in cells of lymphoid origin, this gene or protein, and antibodies to this protein, may show utility as tumor markers and / or immunotherapeutic targets for the tissues listed above. Therefore, this also means arthritis, asthma, immunodeficiency diseases (
For example, it can be used as a drug for immunological disorders including AIDS), leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, pressure ulcers, and psoriasis. In addition, the gene product may have commercial utility in the expansion of various blood lineage stem cells and directed precursors, as well as in the growth of various cell types. Furthermore, translation products corresponding to this gene, as well as antibodies against these translation products,
It may show utility as a tumor marker and / or immunotherapy target for the tissues listed above.

(Characteristics of protein encoded by gene number 10)
The gene encoding the disclosed cDNA is believed to be present on chromosome 19. Therefore, the polynucleotide relating to the present invention is useful as a marker in linkage analysis for chromosome 19.

This gene is predominantly in T-cell helpers and testis, and to a lesser extent ubiquitously.
Expressed in

The polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample, and the following: diseases including but not limited to testicular and immune system diseases and / or disorders And useful as a reagent for the diagnosis of conditions. Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For many disorders of the tissue or cells, especially testis and immune system, significantly higher than the standard gene expression level (ie, the level of expression in healthy tissue or fluid from an individual who does not have the disorder), Alternatively, lower levels of expression of this gene may result in specific tissues or cell types (eg, testicular tissue, immune tissue, cancerous tissue and wound tissue) or body fluids (eg, lymphatic tissue) taken from individuals with such disorders. , Serum, plasma, urine, synovial fluid and cerebrospinal fluid), or in another tissue or sample.

The observed tissue distribution suggests that this gene product may be useful in the diagnosis and / or treatment of various disorders. Based on tissue distribution in helper T cells, this gene product may be useful in the diagnosis and / or treatment of immune system dysfunction, leukemia / lymphoma, susceptibility to infection, and circulatory disorders. Similarly, increased expression in the testis suggests a potential role in male fertilization and a potential application of this gene product as a male contraceptive. Secreted proteins also identify agents that modulate their interactions to isolate cognate ligands or receptors, as tissue markers, to elicit antibodies, to determine biological activity And can be used as a nutritional supplement. This product also has a very wide range of biological activities. These representatives are:
Induction of cytokines, cell proliferation / differentiation regulating activity, or other cytokines; immunostimulatory / immunosuppressive activity (eg to treat human immunodeficiency virus infection, cancer, autoimmune diseases, and allergies); regulation of hematopoiesis (Eg to treat anemia or as an adjunct to chemotherapy); bone, cartilage, tendon, ligament, and / or
Or nerve stimulation or proliferation (eg to treat wounds, to stimulate follicle stimulating hormone (to control fertility); chemotaxis and chemokinesis activity (eg to treat infections, tumors); hemostasis Or thrombolytic activity (eg to treat hemophilia, myocardial infarction, etc.); anti-inflammatory activity (eg to treat septic shock, Crohn's disease); as an antibacterial agent; psoriasis or other hyperproliferative diseases For the regulation of metabolism and behavior, the use of corresponding nucleic acids in gene therapy procedures is also contemplated, and the translation products corresponding to this gene, as well as antibodies to these translation products, are described above. It may show utility as a tumor marker and / or immunotherapy target for the listed tissues.

(Characteristics of protein encoded by gene number 11)
This gene is mainly expressed in the following tissues / cDNA libraries:
prostate, Dendritic cells, pooled; Soar
es_fetal_long_NbHL19W, and to a lesser extent: Human Pine Gland; L428, and Active
aated T-Cell (12hs) / Thiolidene label
edEco.

The polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample, and include but are not limited to: diseases and / or disorders of the immune system, and cancer It is useful as a reagent for diagnosis of diseases and conditions. Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For many disorders of the tissue or cells, especially the immune system and cancerous tissue, significantly higher than the standard gene expression level (ie, the level of expression in healthy tissue or body fluid from an individual who does not have the disorder) Or a lower level of expression of this gene may result in certain tissues or cell types (eg, immune, cancerous and wounded tissues) or body fluids (eg, lymph, Serum, plasma, urine, synovial fluid and cerebrospinal fluid), or in another tissue or sample.

Tissue distribution in human prostate and hematopoietic cell lines indicates that this gene and its protein product are useful for the diagnosis and treatment of human prostate cancer and immune system disorders. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes, and may also suggest utility in the treatment of cancer (eg, by boosting the immune response). Since this gene is expressed in cells of lymphoid origin, this gene or protein, and antibodies to this protein, may show utility as tumor markers and / or immunotherapy targets for the tissues listed above. Thus, it also includes arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, psoriasis, also useful as an agent for immunological disorders obtain. In addition, the gene product may have commercial utility in the expansion of various blood lineage stem cells and directed progenitor cells, and in the differentiation and / or proliferation of various cell types. Furthermore, the translation products corresponding to this gene, and antibodies against these translation products may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

  (Characteristics of the protein encoded by gene no. 12) This gene is mainly expressed in the following tissues / cDNA libraries: Human Whole Brain # 2-OligodT> 1.5 Kb; Keratinocyte and to a lesser extent In the following: Bone Marlow Cell Line (RS4, 11); T cell helper II; Testis 1; remake; Human Stomach, re-exclusion; Lymph node breast Cancer; Human Infant Brain; Breast Cancer Cell line, angiogenic; Human Activated T-Cells; T-Cell PHA 24 hrs; Stratagene hNT neuron (# 937233); Human Testes Tumor; Primary Dendritic cells, frac 2; Human Adult Pulmonary, re-exclusion; Human 8 Week Where Embryo; Soares_fetal_lung_NbHL19W, and Primary Dendritic Cells, lib 1.

The polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample, and include, but are not limited to: diseases and / or disorders of the nervous system and immune system It is useful as a reagent for diagnosis of diseases and conditions. Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. Is it significantly higher than the standard gene expression level (ie, the level of expression in healthy tissue or body fluid from an individual who does not have the disorder) for many disorders of the tissue or cells, particularly the nervous and immune systems? Or a lower level of expression of this gene indicates that certain tissues or cell types (eg, neural tissue, immune tissue, cancerous tissue and wound tissue) or body fluids (eg, Lymph, serum, plasma, urine, synovial fluid and spinal fluid), or in another tissue or sample, can be routinely detected.

The tissue distribution of this gene suggests that this clone may be useful for the diagnosis and / or treatment of neurogenesis, neurodegeneration, hematopoiesis and immune disorders and neoplasms. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes, which may suggest utility in the treatment of cancer (eg, by boosting the immune response). Since this gene is expressed in cells of lymphoid origin, this gene or protein, and antibodies to this protein, may show utility as tumor markers and / or immunotherapeutic targets for the tissues listed above. Thus, it is also used as a drug for immunological disorders including arthritis, asthma, immunodeficiency diseases (eg AIDS), leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, pressure ulcers, and psoriasis. obtain. further,
This gene product may have commercial utility in the expansion of various blood lineage stem cells and directed precursors, as well as in the growth of various cell types. Furthermore, the translation products corresponding to this gene, and antibodies against these translation products may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of protein encoded by gene number 13)
When tested against the U937 bone marrow cell line, supernatants obtained from cells containing this gene activated the GAS assay. Therefore, this gene is Jak-ST
It appears to activate bone marrow cells via the AT signaling pathway. The gamma activating sequence (GAS) is a promoter element found upstream of many genes involved in the Jak-STAT pathway. The Jak-STAT pathway is a large signal transduction pathway involved in cell differentiation and proliferation. Therefore, Jak-STA
Activation of the T pathway (reflected by GAS element binding) can be used to indicate proteins involved in cell proliferation and differentiation.

This gene is ubiquitously expressed with some enrichment in neural / sensory tissues and in primary dendritic cells.

The polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample, and include, but are not limited to: diseases and / or disorders of the nervous system and immune system It is useful as a reagent for diagnosis of diseases and conditions. Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. Is it significantly higher than the standard gene expression level (ie, the level of expression in healthy tissue or body fluid from an individual who does not have the disorder) for many disorders of the tissue or cells, particularly the nervous and immune systems? Or a lower level of expression of this gene indicates that certain tissues or cell types (eg, neural tissue, immune tissue, cancerous tissue and wound tissue) or body fluids (eg, Lymph, serum, plasma, urine, synovial fluid and spinal fluid), or in another tissue or sample, can be routinely detected.

The observed tissue distribution suggests utility for the study, diagnosis and / or treatment of various diseases. Enriched expression in nerve / sensory tissue indicates that this gene may be useful for the treatment, detection and / or diagnosis of neurodegenerative and behavioral disorders such as Alzheimer's disease, Parkinson's disease, learning disorders. Alternatively, expression in dendritic cells and observed biological activity suggests utility for the study, diagnosis, and / or treatment of immune system diseases and / or disorders and infectious diseases. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes, suggesting utility in the treatment of cancer (eg, by boosting the immune response). Since this gene is expressed in cells of lymphoid origin, this gene or protein, and antibodies to this protein, may show utility as tumor markers and / or immunotherapy targets for the tissues listed above. Thus, this gene is also used as a drug for immunological disorders including arthritis, asthma, immunodeficiency diseases (eg AIDS), leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, pressure ulcers and psoriasis Can be. Furthermore, the gene products may have commercial utility in the expansion of various blood lineage stem cells and directed precursors, and in the differentiation and / or proliferation of various cell types. Furthermore, the translation products corresponding to this gene, and antibodies against these translation products may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of protein encoded by gene number 14)
This gene is expressed primarily in immune / hematopoietic cells, especially monocytes and dendritic cells, and fetal liver and spleen (known to have hematopoietic function).

The polynucleotides and polypeptides of the invention are for differential identification of tissues or cell types present in a biological sample, and the following: diseases and conditions including but not limited to diseases and / or disorders of the immune system It is useful as a reagent for diagnosis of Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. Regarding many disorders of the above tissues or cells, especially hematopoietic and developmental systems,
A significantly higher or lower level of expression of this gene relative to a standard gene expression level (ie, an expression level in healthy tissue or body fluid from an individual who does not have the disorder) A particular tissue or cell type (eg, immune system, cancerous tissue and wound tissue) or body fluid (eg, lymph, serum, plasma, urine, synovial fluid and spinal fluid) taken from an individual having, or another tissue or It can be routinely detected in the sample.

Its expression in fetal liver and spleen, known to have hematopoietic function,
And based on expression in dendritic cells, this gene and the polypeptide it encodes are associated with excessive activity of the immune system as observed in autoimmune disorders, including systemic lupus erythematosus (SLE) and rheumatoid arthritis, etc. May be useful in the diagnosis and treatment of disorders associated with immune function, or in the treatment of disorders associated with poor immune function, such as certain immune cell cytopenias. This gene product may also be useful in modulating immune responses in organ transplants and allergies.
This gene may be useful in the treatment of immune disorders or in the initiation of immune-based therapy against tumors. Furthermore, the translation products corresponding to this gene, and antibodies against these translation products, are tumor markers for the tissues listed above and / or
Or it may show utility as a target for immunotherapy.

(Characteristics of protein encoded by gene number 15)
Using the computer algorithm BLASTX, the translation product of this gene is, as a non-limiting example, the accession number gb | AAB5767 in the following database:
9.1 | A sequence accessible through all the information available through the accession number listed (herein incorporated by reference)
ransembrane receptor UNC5H2 [Rattus
norvegicus] ”) and shared sequence homology. A partial alignment showing the observed homology is shown immediately below.

上記で「Ｓ」として示されるｇｂ｜ＡＡＢ５７６７９．１｜のセグメントは、
配列番号：として配列表中に示される。構造的な類似性に基づいて、これらの相
同なポリペプチドは、少なくともいくつかの生物学的活性を共有することが予期
される。このような活性は、当該分野で公知であり、そしてそのいくつかが本明
細書中の他で記載される。このような活性を決定するためのアッセイもまた、当
該分野で公知であり、そのいくつかが、本明細書中の他で記載されている。本発
明の好ましいポリペプチドは、上記で示されるアライメント（コンピューターに
よって、配列に導入されたギャップは、もちろん除去される）において、「Ｑ」
配列に対応する配列番号：として配列表に示されるアミノ酸配列を有するポリペ
プチドを含む。

The segment of gb | AAB57679.1 |, denoted as “S” above, is
It is shown in the sequence listing as SEQ ID NO :. Based on structural similarity, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art and some are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein. Preferred polypeptides of the invention have a “Q” in the alignment shown above (gap introduced into the sequence by the computer is of course removed).
A polypeptide having the amino acid sequence shown in the sequence listing as SEQ ID NO: corresponding to the sequence is included.

The translation product of this gene shares sequence homology with the netrin receptor, which is thought to be important in the developing nervous system. This netrin is
Phylogenetically conserved guidance cues (guidance cu: gu) that can function as diffusive attractants and as repellents for different classes of cells and axons
family of idence cue). In vertebrates, insects and nematodes, members of the DCC subfamily of the immunoglobulin superfamily are:
It is regarded as a receptor involved in migration towards the netrin source. Based on the sequence similarity of the human gene product to the rat netrin receptor, the translation product of this clone is expected to share at least some biological activity with the netrin receptor protein. Such activities are known in the art,
Some are described elsewhere in this specification.

In certain embodiments, a polypeptide of the invention has the following amino acid sequence: E
GEGQIFQLHTTLAETPAGSLDTLCSAPGXTVTTQLG
PYAFKIPLSIRQKICNSLDAPNSRGNDWRMLAQKLS
MDRYLNYFATKASPTGVILDLWEALQQDDGDDLNSLA
SALEEMGKSEMLVAVATDGDC (SEQ ID NO: 152),
Or consist of such an amino acid sequence number. In addition, fragments and variants of these polypeptides (eg, the fragments described herein, at least 80%, 85%, 90%, 95% of these polypeptides)
%, 96%, 97%, 98% or 99% identical polypeptides, and polypeptides encoded by polynucleotides that hybridize under stringent conditions to polynucleotides encoding these polypeptides). Are encompassed by the present invention. Antibodies that bind to the polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

This gene is mainly expressed in the following tissues / cDNA libraries:
normalized infant brain cDNA, and to a lesser extent: Smooth muscle, serum treat
ed; NCI_CGAP_Kid5; Soares_multiple_scl
erosis_2NbHMSP; Soares sovereign tutor Nb
HOT; Soares_pregnant_uterus_NbHPU; E
pididiymus, cauda; Smooth Muscle- HAST
E normalized; Human Synovium; Gessler
Wilms tumor; Human Chondrosarcoma; Hum
an Thymus Stromal Cells; NCI_CGAP_Pan
1; Fetal Heart; NCI_CGAP_GC4; Osteoblas
ts; Human Cerebellum; Soares_NFL_T_GBC
_S1; Soares_NhHMPu_S1; Soares infant b
rain 1NIB; Human Astrocyte; Sinus pini
formis Tumour; Human OB MG63 treated (
10 nM E2) fraction I; Barstead spleen
HPRB2; Normal Human Trabecular Bone
Cells; Adiposites, re-exclusion; Smooth
Muscle Serum Treated, Norm; Smooth mus
cle-ILb induced; Human Normal Breast;
NCI_CGAP_AA1; Plate normal; Human adu
lt (K. Okbo); Kidney Cortex, subtract
ed; Human Osteosarcoma; Prostate BPH; S
pinal Cord, re-exclusion; Breast Cancer
Cell line, angiogenic; 12 Week Old Ea
rly Stage Human, II; Human Osteoblasts
II; Human Pancreas Tumor; Strom cel
l TF274; Human Pancreas Tumor, Reexcis
ion; Overy, Cancer (9809C332): Poorly di
fermented adenocarcinoma; Hemangio
pericytoma; Soares breast 2NbHBst; Hum
an Adrenal Gland Tumor; Smooth muscle
, Serum induced, re-exc; Pancreas Islet
Cell Tumor; Colon Normal II; Adipocyt
es; NCI_CGAP_Co8; Human Fetal Kidney, R
eexcision; NCI_CGAP_Kid3; Human Bone M
arrow, treated; Hodgkin's Lymphoma II;
Keratinocyte; Soares_fetal_long_NbHL1
9W, and Colon Tumor II.

The polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample, and the following: diseases and conditions, including but not limited to nervous system diseases and / or disorders It is useful as a reagent for diagnosis of Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For many disorders of the tissue or cell, particularly the nervous system, significantly higher than or above the standard gene expression level (ie, the level of expression in healthy tissue or fluid from an individual who does not have that disorder) A low level of expression of this gene indicates that certain tissues or cell types (eg, neural tissue, cancerous tissue and wound tissue) or body fluids (eg, lymph,
Serum, plasma, urine, synovial fluid and cerebrospinal fluid), or another tissue or sample,
It can be detected routinely.

Tissue distribution in brain and transmembrane receptor UNC5H2 [Rattus n
orvegicus] indicates that the protein product of this clone can detect, treat and / or detect neurodegenerative disease states, behavioral disorders, or inflammatory conditions.
Or it is useful for prevention. Typical applications include the following “
In the sections "Regeneration" and "Hyperproliferative disorders" are described in Examples 11, 15 and 18 and any other. In short, this application includes Alzheimer's disease, Parkinson's disease, Huntington's disease, Tourette syndrome, meningitis, encephalitis, demyelinating disease, peripheral neuropathy, neoplasia, trauma, congenital abnormalities,
Spinal cord injury, ischemia and infarction, aneurysm, bleeding, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disorder, ALS, psychosis, autism, and behavioral changes (nutrition Detection, including supplementation, sleep patterns, balance, and sensory disturbances)
Treatment, and / or prophylaxis include but are not limited to. Furthermore, increased expression of this gene product in the brain region indicates that it plays a role in normal neural function. Potentially, this gene product is involved in synaptogenesis, neurotransmission, learning, recognition, homeostasis, or neuronal differentiation or survival. Furthermore, in addition to its use as a nutritional supplement, this protein is also used to elicit antibodies, to isolate cognate ligands or receptors as tissue markers, to determine biological activity. Can be used to identify agents that modulate their interaction. The protein and antibodies against this protein may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of protein encoded by gene number 16)
This gene is mainly expressed in the following tissues / cDNA libraries:
Dendritic cells, pooled; human tons
.

The polynucleotides and polypeptides of the invention are for differential identification of tissues or cell types present in a biological sample, and the following: diseases and conditions including but not limited to diseases and / or disorders of the immune system It is useful as a reagent for diagnosis of Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For a number of disorders of the tissue or cells, particularly the immune system, significantly higher or higher than the standard gene expression level (ie, the level of expression in healthy tissue or body fluid from an individual without the disorder) A low level of expression of this gene indicates that certain tissues or cell types (eg, immune tissue, cancerous tissue and wound tissue) or body fluids (eg, lymph,
Serum, plasma, urine, synovial fluid and cerebrospinal fluid), or another tissue or sample,
It can be detected routinely.

Based on its expression in tonsils and dendritic cells, this gene and its encoded polypeptide are found in excess of the immune system as observed in autoimmune disorders, including systemic lupus erythematosus (SLE) and rheumatoid arthritis. It may be useful in the diagnosis and treatment of disorders associated with immune function such as activity, or in the treatment of disorders associated with poor immune function such as certain immune cell cytopenias. This gene product may also be useful in modulating immune responses in organ transplants and allergies.

(Characteristics of protein encoded by gene number 17)
Preferred polypeptides of the present invention include or consist of a polypeptide having the amino acid sequence shown as SEQ ID NO: 148 in the sequence listing. In addition, fragments and variants of these polypeptides (eg,
The fragments described herein, at least 80% of these polypeptides,
Encoded by polypeptides that are 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, and polynucleotides that hybridize under stringent conditions to polynucleotides encoding these polypeptides Polypeptides, etc.) are also encompassed by the present invention. Antibodies that bind to the polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

This gene is mainly expressed in the following tissues / cDNA libraries:
KMH2; Human Adrenal Gland Tumor; Dendr
itic cells, pooled; Bone Marlow Cell L
ine (RS4, 11).

The polynucleotides and polypeptides of the invention are for differential identification of tissues or cell types present in a biological sample, and the following: diseases and conditions including but not limited to diseases and / or disorders of the immune system It is useful as a reagent for diagnosis of Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For a number of disorders of the tissue or cells, particularly the immune system, significantly higher or higher than the standard gene expression level (ie, the level of expression in healthy tissue or body fluid from an individual without the disorder) A low level of expression of this gene indicates that certain tissues or cell types (eg, immune tissue, cancerous tissue and wound tissue) or body fluids (eg, lymph,
Serum, plasma, urine, synovial fluid and cerebrospinal fluid), or another tissue or sample,
It can be detected routinely.

Based on expression in bone marrow and dendritic cells, this gene and its encoded gene product are immune immunity including autoimmune syndromes such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, diabetes, and immunodeficiency syndrome May be useful in the diagnosis and treatment of diseases of the system. This gene may also be useful for modulating the immune response in conditions such as organ transplantation or allergies. Furthermore, the translation products corresponding to this gene, and antibodies against these translation products may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of protein encoded by gene number 18)
This gene is mainly expressed in two different types of tissues, such as immune / hematopoietic cells and testis, as exemplified in the following tissues / cDNA libraries: Human Eosinofils; Human T
estes, Reexcision; NCI_CGAP_Pr3; Early
Stage Human Brain; Endothelial-induce
d; Activated T-cell (12h) / Thiouridine-
re-excition; T cell helper II; Soares_
fetal_heart_NbHH19W; Primary Dendriti
c Cells, lib 1; HSC172 cells; Epididi
ymus, caput &corpus; Smooth Muscle- H
ASTE normallyized; Amionic Cells-Pri
mary Culture; NCI_CGAP_Pr22; Soares_pi
near_gland_N3HPG; NCI_CGAP_Pr28; NCI_C
GAP_CLL1; Resting T-Cell Library, II; S
tratagene colon (# 937204); Dendritic c
cells, pooled; Soares_pregnant_uterus_N
bHPU; Soares_NhHMPU_S1; NCI_CGAP_Co1; C
homosome 7 HeLa cDNA Library; Atrium
cDNA library Human heart; Chromome
7 Fetal Brain cDNA Library; Human OB
MG63 control fraction I; Cem cells c
yclohexamide treated; Storm cancer (
human), re-exclusion; Human endometric
stromal cells-treated with estradio
l; Smooth muscle, IL1b induced; Synovia
l hypoxia-RSF subtracted; Human Whore
Brain # 2-Oligo dT> 1.5 Kb; HL-60, P
MA 4H, re-exclusion; NLCAP procel cell
l line; Synovial hypoxia; Jurkat T-cel
l G1 phase; Brain Frontal Cortex, re-e
xcision; Stratagene neuroepithelium (#
937231); Human Umbilical Vein, Reexcis
ion; Gessler Wilms tumor; HUMAN JURKAT
MEMBRANE BOUND POLYSOMES; Human Feta
l Dura Mater; Human Activated T-Cells
; Human Hypothalmus, Schizophrenia; NCI
_CGAP_Br2; Human Rhabdomyosarcoma; Hum
an adult tests, large inserts; Pancre
as Islet Cell Tumor; Colon Carcinoma;
breast lymph node cDNA library; Adipo
cytes; CD34 depleted Buffy Coat (Cord
Blood), re-exclusion; Human Microvascul
ar Endothelial Cells, fract. A; Monocyt
e activated; HUMAN B CELL LYMPHOMA; Sp
leen, Chronic lymphotropic leukemia; Hu
man Bone Marrow, treated; Human Tests
; Soares_fetal_liver_screen_1NFLS_S1,
And Soares live river screen 1NFLS.

The polynucleotides and polypeptides of the invention are for differential identification of tissues or cell types present in a biological sample, and the following: diseases and conditions including but not limited to diseases and / or disorders of the immune system It is useful as a reagent for diagnosis of Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For a number of disorders of the tissue or cells, particularly the immune system, significantly higher or higher than the standard gene expression level (ie, the level of expression in healthy tissue or body fluid from an individual without the disorder) A low level of expression of this gene indicates that certain tissues or cell types (eg, immune tissue, cancerous tissue and wound tissue) or body fluids (eg, lymph,
Serum, plasma, urine, synovial fluid and cerebrospinal fluid), or another tissue or sample,
It can be detected routinely.

Based on its expression in various sources related to immune function (eg, eosinophils, lymph nodes, T cells and dendritic cells), this gene and its encoded polypeptide have been linked to autoimmune syndrome (systemic lupus erythematosus). For the diagnosis and treatment of disorders related to immune function, such as those observed in (SLE) and rheumatoid arthritis), such as disorders in which excessive activity of the immune system is pathogenic, or for specific immune cell depletion It may be useful in the treatment of disorders associated with poor immune function such as disease. This gene may also be useful for modulating the immune response in conditions such as organ transplantation or allergies. Furthermore, the translation products corresponding to this gene, and antibodies against these translation products may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of protein encoded by gene number 19)
This gene is mainly expressed in the following tissues / cDNA libraries:
Human Fetal Brain; pBMC simulated w /
poly I / C.

The polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample, and the following: diseases and conditions, including but not limited to nervous system diseases and / or disorders It is useful as a reagent for diagnosis of Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For many disorders of the tissue or cell, particularly the nervous system, significantly higher than or above the standard gene expression level (ie, the level of expression in healthy tissue or fluid from an individual who does not have that disorder) A low level of expression of this gene indicates that certain tissues or cell types (eg, neural tissue, cancerous tissue and wound tissue) or body fluids (eg, lymph,
Serum, plasma, urine, synovial fluid and cerebrospinal fluid), or another tissue or sample,
It can be detected routinely.

Due to expression in the developing brain, this gene and its encoded polypeptide are used to diagnose and treat stroke, Alzheimer's disease, multiple sclerosis, ALS, and other disorders associated with loss of cognitive and learning functions. Can be useful to. This protein may also be useful in the treatment of spinal cord and other nerve injuries that require nerve cell regeneration and regrowth. Furthermore, the translation product corresponding to this gene,
In addition, antibodies against these translation products may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of protein encoded by gene number 20)
This gene is mainly expressed in the following tissues / cDNA libraries:
Soares_pregnant_uterus_NbHPU, and to a lesser extent: Human endometrical
cells-treated with progesterone; NCI
_CGAP_Pr28; Human endometrical stromal
cells; Keratinocyte; Human Who Brai
n # 2-Oligo dT> 1.5 Kb; Soares_NhHMP
u_S1; NCI_CGAP_GCB1; Human endometric
stromal cells-treated with estradio
l; NCI_CGAP_Co10; NCI_CGAP_Ut2; NCI_CGA
P_Gas4; NCI_CGAP_Pan1; NCI_CGAP_GC4; NC
I_CGAP_GC6; Soares overtime tutor NbHOT;
Bone Marlow Cell Line (RS4, 11); Hodgki
n's Lymphoma II; Human 8 Week Where E
mbryo; Soares_fetal_lung_NbHL19W; T ce
ll helper II; Soares_NFL_T_GBC_S1; Hea
ling Abdomen Wound, 15 days post inci
sion; NCI_CGAP_Ov35; Testis 1; Human 8
Week Where Embryo, subtracted; Human r
etina cDNA Tsp509I-cleaved subunit
y; NCI_CGAP_Ov23; Activated T-cells;
Epididymus, cauda; Stratagene schizo
brain S11; NCI_CGAP_Co9; Human Umbic
al Vein, Endo. remake; Overy, Cancer: (15
799A1F) Poorly differentiated carcino
ma; Human Stomach, re-exclusion; Wilm's
human; Manic Depression Tissue;
H. Lymph node breast cancer; Human Inf
ant Brain; Breast Cancer Cell line, an
geogenic; NCI_CGAP_Ut1; NCI_CGAP_Kid6;
Human Fetal Dura Mater; Human Activat
ed T-Cells; T-Cell PHA 24 hrs; NCI_CGA
P_CLL1; Human Thymus Stall Cells; CH
ME Cell Line, treated 5 hrs; Stratagen
e hNT neuron (# 937233); Human T-Cell L
ymphoma; Colon Carcinoma; Human Placen
ta; NCI_CGAP_Kid11; Human Tests Tumor
; Primary Dendritic cells, frac 2; Panc
reas normal PCA4 No; Human Adult Pulm
only, re-exclusion; Human Bone Marrow,
treated; Soares_parathyroid_tumor_NbH
PA; Soares melanocite 2NbHM; Colon Tum
or II; Soares_fetal_heart_NbHH19W, and Primary Dendritic Cells, lib 1.

The polynucleotides and polypeptides of the invention include for the differential identification of tissues or cell types present in a biological sample and include the following: reproductive, immune, and hematopoietic diseases and / or disorders Useful as reagents for the diagnosis of non-limiting diseases and conditions. Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. Significant over standard gene expression levels (ie, expression levels in healthy tissues or body fluids from individuals who do not have the disorder) for many disorders of the tissues or cells, particularly the reproductive, immune and hematopoietic systems Higher or lower levels of expression of this gene, such as certain tissues or cell types taken from individuals with such disorders (eg, reproductive tissue, immune tissue, hematopoietic tissue, cancerous tissue and wound tissue) ) Or body fluids (eg, lymph, serum, plasma, urine, synovial fluid and cerebrospinal fluid), or in another tissue or sample.

The observed tissue distribution suggests that this gene product may be useful in the diagnosis and / or treatment of diseases of the reproductive system, neoplasia, and other proliferative disorders. Similarly, expression in hematopoietic cells is responsible for the role in proliferation, survival, differentiation and / or activation of various blood cell lineages, as well as this gene in immune dysfunction, autoimmunity, leukemia / lymphoma, susceptibility to infection, and circulatory disorders It may indicate product involvement. Furthermore, the translation products corresponding to this gene, and antibodies against these translation products may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of protein encoded by gene number 21)
Preferred polypeptides of the present invention comprise or consist of a polypeptide having the amino acid sequence 157) shown as SEQ ID NO: SEQ ID NO: and / or SEQ ID NO: 156 in the sequence listing. In addition, fragments and variants of these polypeptides (eg, the fragments described herein, at least 80%, 85%, 90%, 95%, 96
Polypeptides that are%, 97%, 98% or 99% identical, and polypeptides encoded by polynucleotides that hybridize under stringent conditions to polynucleotides encoding these polypeptides, etc.)
Included by the present invention. Antibodies that bind to the polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

The translation product corresponding to this gene shares sequence homology with human dermatan / chondroitin sulfate 2-sulfotransferase (see, eg, Genbank accession number AB020316).

This gene is mainly expressed in the following tissues / cDNA libraries:
STROMAL -OSTEOCLASTOMA; Dendritic cel
ls, pooled; Smooth muscle, control; Nine
Week Old Early Stage Human.

The polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample, and the following: diseases including but not limited to diseases and / or disorders of musculoskeletal tissues And useful as a reagent for the diagnosis of conditions. Similarly, polypeptides and antibodies to these polypeptides are useful for providing immunological probes for differential identification of tissues or cell types. For many disorders of the tissue or cells, particularly the musculoskeletal system, significantly higher than the standard gene expression level (ie, the level of expression in healthy tissue or fluid from an individual without the disorder), or Lower levels of expression of this gene indicate that certain tissues or cell types (eg, musculoskeletal tissue, cancerous tissue and wound tissue) or body fluids (eg, lymph, serum, In plasma, urine, synovial fluid and cerebrospinal fluid), or in another tissue or sample.

This secreted protein elicits antibodies to determine biological activity,
It can be used as a tissue marker, to isolate homologous ligands or receptors, to identify agents that modulate their interaction, and as a nutritional supplement. The protein can also have a very wide range of biological activities. Typical applications are the following “chemotaxis” and “binding activity” sections, Examples 11, 12, 1
3, 14, 15, 16, 18, 19 and 20 and elsewhere herein. Briefly, the protein may have the following activities: cytokines, cell growth / differentiation regulating activity, or induction of other cytokines; immunostimulatory / immunosuppressive activity (eg, human immunodeficiency virus infection, cancer Regulation of hematopoiesis (eg to treat anemia or as an adjunct to chemotherapy); stimulation or proliferation of bones, cartilage, tendons, ligaments, and / or nerves; (Eg, for treating wounds, for stimulation of follicle stimulating hormone (for control of fertility); chemotaxis and chemokinesis activity (eg,
Anti-inflammatory activity (eg to treat septic shock, Crohn's disease); antibacterial drugs As; to treat psoriasis or other hyperproliferative diseases; to regulate metabolism and behavior. The use of corresponding nucleic acids in gene therapy procedures is also contemplated. The increased level of expression of this gene product in giant cell tumors
This suggests that this product may play a role in the survival, proliferation and / or growth of osteoclastoma. Thus, this product may be useful to affect bone mass in conditions such as osteoporosis. In addition, in addition to its use as a nutritional supplement, this protein can also be used to determine biological activity, elicit antibodies, as a tissue marker, to isolate homologous ligands or receptors. Can be used to identify agents that modulate the interaction. The protein and antibodies against this protein may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

表１は、上記の各「遺伝子番号」に対応する情報を要約する。「ヌクレオチド
配列番号Ｘ（ＮＴ ＳＥＱ ＩＤ ＮＯ：Ｘ）」として同定されるヌクレオチド
配列は、表１において同定される「ｃＤＮＡクローンＩＤ」から、およびいくつ
かの場合において、さらなる関連のＤＮＡクローンから得られる部分的に相同な
（「重複する」）配列から構築された。重複する配列は、高い重複性の単一の連
続した配列に構築され（通常、各ヌクレオチド部位で３〜５個の重複する配列）
、配列番号Ｘ（ＳＥＱ ＩＤ ＮＯ：Ｘ）として同定される最終的な配列を得た
。

Table 1 summarizes the information corresponding to each of the above “gene numbers”. The nucleotide sequence identified as “NT SEQ ID NO: X” is obtained from the “cDNA clone ID” identified in Table 1 and, in some cases, from further related DNA clones. Constructed from partially homologous ("overlapping") sequences. Overlapping sequences are built into a single contiguous sequence with high redundancy (usually 3-5 overlapping sequences at each nucleotide site)
The final sequence identified as SEQ ID NO: (SEQ ID NO: X) was obtained.

The cDNA clone ID is deposited on that date, and “ATCC accession number Z
And the corresponding accession numbers listed in “Date”. Some of the deposits contain multiple different clones corresponding to the same gene. “Vector” refers to the type of vector contained in the cDNA clone ID.

The “total nucleotide sequence (Total NT Seq)” is the “gene number (G
ene No) "refers to the total number of nucleotides in the contig. The deposited clone may contain all or most of these sequences, which are indicated as “5 ′ nucleotides of the cloned sequence” and “3 ′ nucleotides of the cloned sequence” of SEQ ID NO: X. This is reflected by the position of the nucleotide to be selected. Presumed start codon (methionine) SEQ ID NO: SEQ
The nucleotide position of ID NO: X) is identified as “the 5 ′ nucleotide of the start codon”. Similarly, SEQ ID NO: SEQ ID NO (SEQ ID
The nucleotide position of NO: X) is “5 of the first amino acid of the signal peptide.
Identified as 'nucleotide'.

The translated amino acid sequence begins with methionine and is “amino acid sequence number Y (S
Although identified as "EQ ID NO: Y)", other reading frames can also be readily translated using known molecular biology techniques. Polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.

The positions of the first and last amino acids of the putative signal peptide SEQ ID NO: Y (SEQ ID NO: Y) are identified as “the first amino acid of the signal peptide” and “the last amino acid of the signal peptide”. SEQ ID NO: Y (S
The position of the putative first amino acid of EQ ID NO: Y) is identified as “the putative first amino acid of the secretory part”. Finally, the amino acid position of SEQ ID NO: Y of the last amino acid in the open reading frame is identified as “the last amino acid of the ORF”.

SEQ ID NO: (SEQ ID NO: X) (where X can be any polynucleotide sequence disclosed in the sequence listing) and translated SEQ ID NO: Y (SEQ
ID NO: Y) (where Y can be any polypeptide sequence disclosed in the sequence listing) is sufficiently accurate, and otherwise various uses well known in the art and Are suitable for various applications described further in. For example, SEQ ID NO: (SEQ ID NO: X)
: X) nucleic acid sequence contained in or cDNA contained in the deposited clone
It is useful for designing a nucleic acid hybridization probe that detects.
These probes also hybridize to nucleic acid molecules in the biological sample,
This allows various forensic and diagnostic methods of the present invention. Similarly, the polypeptide identified from SEQ ID NO: Y is specific for, for example, the proteins (including polypeptides and secreted proteins) encoded by the cDNA clones identified in Table 1. It can be used to make antibodies that bind.

Nevertheless, the DNA sequence produced by the sequencing reaction may contain sequencing errors. This error is a misidentified nucleotide
Or as an insertion or deletion of nucleotides in the generated DNA sequence. Incorrectly inserted or deleted nucleotides cause a frameshift in the reading frame of the deduced amino acid sequence. In these cases, even if the DNA sequence produced can be more than 99.9% identical to the actual DNA sequence (eg, a single base insertion or deletion in an open reading frame of more than 1000 bases) The deduced amino acid sequence is different from the actual amino acid sequence.

Thus, for those applications that require accuracy in nucleotide or amino acid sequences, the present invention relates to a generated nucleotide sequence identified as SEQ ID NO: X (SEQ ID NO: X), and SEQ ID NO: Y ( SEQ ID N
Also provided is a sample of plasmid DNA containing the human cDNA of the present invention deposited with the ATCC, as described in Table 1, as well as the putative translated amino acid sequence identified as O: Y). The nucleotide sequence of each deposited clone can be readily determined by sequencing the deposited clone according to known methods. The deduced amino acid sequence can then be verified from such deposits. In addition, the amino acid sequence of the protein encoded by a particular clone can also be expressed by peptide sequencing or in a suitable host cell containing the deposited human cDNA, the protein collected and the sequence By determining it can be determined directly.

The present invention also includes SEQ ID NO: (SEQ ID NO: X), SEQ ID NO: Y (SEQ
ID NO: Y), or the gene corresponding to the deposited clone. Corresponding genes can be isolated according to known methods using the sequence information disclosed herein. Such methods include the steps of preparing probes or primers from the disclosed sequences and identifying or amplifying the corresponding gene from an appropriate source of genomic material.

Also provided in the present invention are allelic variants, orthologs (or
tholog), and / or species homologs. Using procedures known in the art, using the sequences disclosed herein or information from clones deposited with ATCC, SEQ ID NO: (SEQ ID NO: X), SEQ ID NO: Y (S
EQ ID NO: Y) or the full-length gene, allelic variant, splicing variant, full-length coding portion, ortholog, and / or species homolog of the gene corresponding to the deposited clone may be obtained. For example, allelic variants and / or
Or species homologs are isolated and identified by making appropriate probes or primers from the sequences provided herein and screening appropriate nucleic acid sources for allelic variants and / or desired homologs. Can be done.

Table 2 shows preferred epitopes included in certain embodiments of the invention, and polypeptide sequences that can be discarded according to certain embodiments of the invention. The first column is
Each “gene number” described in Table 1 above is referred to. The second column shows the sequence identifier “nucleotide sequence number X” for the polynucleotide sequences disclosed in Table 1. 3
The column shows the sequence identifier “AA SEQ ID NO: Y” for the polynucleotide sequences disclosed in Table 1. The fourth column is SEQ ID NO: 1 and the last nucleotide minus 15
A unique integer “ntA”, which is any integer between and. And the fifth column shows a unique integer “ntB”, which is an arbitrary integer between 15 of SEQ ID NO: X and the last nucleotide. Here, both ntA and ntB correspond to the positions of the nucleotide residues shown in SEQ ID NO: X, and ntB is +14 or more. For each polynucleotide shown as SEQ ID NO: X, a uniquely defined integer may be substituted for the general formula ab and may be used to describe a polynucleotide that may preferably be excluded from the present invention. Column 6 lists the residues containing the preferred epitope contained in each preferred ORF encoded polypeptide (SEQ ID NO: Y). The identification of potential immunogenic regions
carried out according to the method of eson and Wolf ((1988) CABIOS, 4; 181-186); in particular, the Genetics C of this algorithm
Implement Group (GCG) (Program PEPTIDESTR)
UCTURE (Wisconsin Package v10.0, Genet
ics Computer Group (GCG), Madison, Wisc
))). This method returns the value of the probability that a given residue is found on the surface of the protein. Regions with an antigenic coefficient score of at least 6 amino acids are shown as “preferred epitopes” in Table 2. A polypeptide of the invention may have 1, 2, 3, 4, 5 or 5 or more of the antigenic epitopes comprising the residues listed in Table 2. Depending on the analytical criteria used, it is understood that the antigenic determinant can be inferred and the exact position of the antigenic determinant can vary slightly.

Table 3 summarizes the expression profiles of the polynucleotides corresponding to the clones disclosed in Table 1. The first column is the cD for each contig sequence disclosed in Table 1.
The specific clone identifier “clone number (Clone) for NA clone
ID) ”. The “library code” in the second column shows the expression profile of the library of tissues and / or cell lines that express the polynucleotide of the invention. The code for each library in the second column indicates the identifier code of the tissue / cell source corresponding to the library code provided in Table 5 and the library description. Expression of these polynucleotides was not observed in other tissues and / or cell libraries tested. Those skilled in the art routinely use this information to identify tissues that exhibit a predominant expression pattern of the corresponding polynucleotide of the invention, or
Alternatively, polynucleotides that exhibit specific tissue expression can be identified.

The first column of Table 4 provides the nucleotide sequence identifier “SEQ ID NO: X”. this"
“SEQ ID NO: X” matches the nucleotide sequence ID X disclosed in column 5 of Table 1. The second column of Table 4 shows the cytological band or chromosome (Cytological Band or Chr) which is the chromosomal arrangement of the polynucleotide corresponding to SEQ ID NO: X.
romosome) ". The chromosome arrangement is NCBI (National
Center for Biotechnology Information
n) Determined by finding exact match to EST and cDNA contained in UniGene database. Considering the estimated chromosomal arrangement, Online Mendlian Inheritance in M
an (Online Mendelian Inheritance in M
an, OMIM ^™ . McKusick-Nathans Institute
for Genetic Medicine, Johns Hopkins U
diversity (Baltimore, MD) and National C
enter for Biotechnology Information,
National Library of Medicine (Bethesd
a, MD) Disease-related loci were determined by comparison with Morbid Map from 2000). World Wide Web URL: http: //
www. ncbi. nlm. nih. gov / omim /). If the estimated chromosome configuration of Query overlaps with the chromosome sequence number position of the Morbid Map entry, the OMIM reference identification number (OMIM r of the morbid map entry)
(reference identity numbers) to OM
“IM ID” is shown in the third column of Table 4. Table 6 shows the keys for the number of OMIM reference identifications.

Table 5 provides clues to the library code disclosed in Table 3.
The first column provides the library code disclosed in the second column of Table 3. The second column provides details of the tissue or cell source from which the corresponding library was derived. The library code corresponding to the diseased tissue is shown in the third column using the term “disease”.

Table 6 provides a clue to the OMIM reference identification number disclosed in the third column of Table 4. The OMIM reference identification number (first column) is Online Mendian
Inheritance in Man (Online Mendelian
Inheritance in Man, OMI M. McKusick-N
atans Institute for Genetic Medicine
e, Johns Hopkins University (Baltimore
, MD) and National Center for Biotechno
logic Information, National Library of
Medicine (Bethesda, MD) 2000) World Wid
e Web URL: http: // www. ncbi. nlm. nih. go
derived from v / omim /). The second column provides the disease associated with the cytoplasmic band disclosed in the second column of Table 4, as determined using the Morbid Map database.

本発明のポリぺプチドは、任意の適切な様式で調製され得る。このようなポリ
ぺプチドとしては、単離された天然に存在するポリぺプチド、組換え的に生成さ
れたポリぺプチド、合成的に生成されたポリぺプチド、またはこれらの方法の組
合せによって生成されたポリぺプチドが挙げられる。このようなポリぺプチドを
調製するための手段は、当該分野において十分に理解される。

The polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or combinations of these methods. Polypeptides that have been prepared. Means for preparing such polypeptides are well understood in the art.

The polypeptide can be in the form of a secreted protein (including the mature form) or can be part of a larger protein (eg, a fusion protein) (see below). It is often advantageous to include additional amino acid sequences, including secretory or leader sequences, pro sequences, sequences that aid purification (eg, multiple histidine residues), or additional sequences for stability during recombinant production. It is.

The polypeptides of the present invention are preferably provided in an isolated form and are preferably substantially purified. Recombinantly generated versions of polypeptides (including secreted polypeptides) can be produced using techniques described herein or other techniques known in the art (eg, Smith and John).
son, Gene 67: 31-40 (1988) as described above). The polypeptide of the present invention also comprises
For example, using the techniques described herein or other techniques known in the art, such as antibodies of the invention raised against secreted proteins, natural sources, synthetic sources or It can be purified from recombinant sources.

The present invention provides a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: X and / or the cDNA contained in ATCC deposit Z.
The present invention also provides the polypeptide sequence of SEQ ID NO: Y and / or ATCC deposit Z
A polypeptide comprising or consisting of a polypeptide encoded by the cDNA contained therein is provided. A polynucleotide encoding a polypeptide comprising or consisting of the polypeptide sequence of SEQ ID NO: Y and / or the polypeptide sequence encoded by the cDNA contained in ATCC deposit Z is also encompassed by the present invention. .

(Signal sequence)
The invention also encompasses a mature form of a polypeptide having the polypeptide sequence of SEQ ID NO: Y and / or the polypeptide sequence encoded by the cDNA in the deposited clone. A polynucleotide encoding the mature form (eg, the polynucleotide sequence of SEQ ID NO: X and / or the cDNA of the deposited clone)
Are also encompassed by the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretory leader sequence that is cleaved from the mature protein once transport of the growing protein chain across the rough endoplasmic reticulum is initiated. . Even most mammalian and insect cells cleave secreted proteins with the same specificity.
However, in some cases, the cleavage of the secreted protein is not completely uniform,
This results in two or more mature species of this protein. Furthermore, it has long been known that the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein (ie, it is unique to the amino acid sequence of the polypeptide).

Methods are available for predicting whether a protein has a signal sequence, as well as a breakpoint for that sequence. For example, McGeoch, Vi
rus Res. 3: 271-286 (1985) uses information from a short N-terminal charged region followed by an uncharged region of the complete (uncut) protein. von Heinje, Nucleic Acids Res
. 14: 4683-4690 (1986) uses information from residues around the cleavage site (typically residues -13 to +2), where +1 indicates the amino terminus of the secreted protein. . The accuracy of predicting the breakpoints of known mammalian secreted proteins for each of these methods is in the range of 75-80% (
von Heinje, supra). However, the two methods do not necessarily produce the same estimated breakpoint for a given protein.

In the present case, the deduced amino acid sequence of the secreted polypeptide is the Signa
a computer program called 1P (Henrik Nielsen et al.,
Protein Engineering 10: 1-6 (1997)), this program predicts the location of proteins in cells based on amino acid sequence. As part of the computer prediction of this localization, McGeoc
The methods of h and von Heinje are incorporated. With this program,
Analysis of the amino acid sequence of the secreted protein described herein provided the results shown in Table 1.

However, as will be appreciated by those skilled in the art, the cleavage site sometimes varies depending on the organism and cannot be predicted with absolute certainty. Thus, the present invention provides a secreted polypeptide having the sequence shown in SEQ ID NO: Y, which has an N-terminus that begins within 5 residues (ie +5 or −5 residues) of the putative breakpoint. Similarly, it is also recognized that in some cases, cleavage of the signal sequence from the secreted protein is not completely uniform, resulting in more than one secreted species. These polypeptides and polynucleotides encoding such polypeptides are contemplated by the present invention.

Furthermore, the signal sequence identified by the above analysis may not necessarily predict a naturally occurring signal sequence. For example, a naturally occurring signal sequence can be further upstream from the predicted signal sequence. However, the putative signal sequence may be able to direct the secreted protein to the ER. Nevertheless,
The present invention relates to a mature protein produced by expression of a polynucleotide sequence of SEQ ID NO: X and / or a polynucleotide sequence contained in the cDNA of a deposited clone in a mammalian cell (for example, COS cell as described below). provide. These polypeptides and polynucleotides encoding such polypeptides are contemplated by the present invention.

(Polynucleotide and polypeptide variants)
The present invention relates to a variant of the cDNA sequence contained in the polynucleotide sequence disclosed in SEQ ID NO: X, its complementary strand, and / or the deposited clone.

The invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID NO: Y and / or the polypeptide sequence encoded by the deposited clone.

“Variant” refers to a polynucleotide or polypeptide that differs from the polynucleotides or polypeptides of the invention but retains their essential properties. In general, variants are generally closely similar and in many regions are identical to a polynucleotide or polypeptide of the invention.

The present invention also includes, for example, a sequence encoding a nucleotide of SEQ ID NO: X or a complementary strand thereof, a sequence encoding a nucleotide contained in a deposited cDNA clone or a complementary strand thereof, and a polypeptide of SEQ ID NO: Y. A nucleotide sequence encoding, a nucleotide sequence encoding a polypeptide encoded by the cDNA contained in the deposited clone, and / or a polynucleotide fragment of any of these nucleic acid molecules (eg, as described herein) Fragment) at least 80%, 85%, 90%, 95%
, 96%, 97%, 98%, or 99% nucleotide sequences that are or consist of nucleotide sequences. Polynucleotides that hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringent conditions, and polypeptides encoded by these polynucleotides, are also encompassed by the present invention.

The invention also includes, for example, the polypeptide sequence shown in SEQ ID NO: Y, the polypeptide sequence encoded by the cDNA contained in the deposited clone, and / or a polypeptide fragment of any of these polypeptides (Eg, fragments described herein) at least 80
%, 85%, 90%, 95%, 96%, 97%, 98%, 99% amino acid sequences comprising or consisting of the same.

For example, by a nucleic acid having a nucleotide sequence that is at least 95% “identical” to a reference nucleotide sequence of the invention, the nucleotide sequence of the nucleic acid is 5 for each 100 nucleotides of the reference nucleotide sequence in which the nucleotide sequence encodes a polypeptide. It is intended to be identical to the reference sequence except that it may contain up to point mutations. In other words, at least 9 relative to the reference nucleotide sequence.
In order to obtain a nucleic acid having a 5% identical nucleotide sequence, up to 5% of the nucleotides of the reference sequence can be deleted or replaced with another nucleotide;
Alternatively, multiple nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. The query sequence can be the entire sequence shown in Table 1, the ORF (open reading frame), or any fragment specified as described herein.

In practice, any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 9% to the nucleotide sequence of the invention.
Whether it is 7%, 98%, or 99% identical can be conventionally determined using known computer programs. A preferred method for determining the best overall match (also referred to as the overall sequence alignment) between the query sequence (sequence of the invention) and the subject sequence is Brutlag et al. (Comp. App
. Biosci. 6: 237-245 (1990)) and can be determined using the FASTDB computer program. In sequence alignment, both the query sequence and the subject sequence are DNA sequences. RNA
The sequences can be compared by converting from U to T. The result of this overall sequence alignment is expressed in percent identity. Preferred parameters used in FASTDB alignment of DNA sequences to calculate percent identity are: Matrix = Unity, k-tuple = 4, Mismatch P
energy = 1, Joining Penalty = 30, Randomiz
ation Group Length = 0, Cutoff Score = 1,
Gap Penalty = 5, Gap Size Penalty 0.05,
Window Size = 500 or the length of the subject nucleotide sequence (whichever is shorter).

If the subject sequence is shorter than the query sequence due to a 5 ′ or 3 ′ deletion (not due to an internal deletion), a manual correction must be made to the result. This is because the FASTDB program does not consider 5 'and 3' cuts of the subject sequence when calculating percent identity. For subject sequences that are cleaved at the 5 'end or 3' end, the percent identity to the query sequence is a query sequence that is 5 'and 3' of the subject sequence that is not matched / aligned as a percentage of the total bases of the query sequence It is corrected by calculating the number of bases. Whether nucleotides are matched / aligned is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated by the FASTDB program described above using specific parameters to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only those bases outside the 5 ′ and 3 ′ bases of the subject sequence that are not matched / aligned with the query sequence, as shown by the FASTDB alignment, are calculated for the purpose of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. Deletions occur at the 5 ′ end of the subject sequence and therefore the FASTDB alignment does not show a match / alignment at the first 10 bases of the 5 ′ end. 1
Zero unpaired bases represent 10% of the sequence (number of bases at unmatched 5 ′ and 3 ′ ends / total number of bases in the query sequence), so 10% is FASTD
Subtracted from the percent identity score calculated by the B program.
If the remaining 90 bases are a perfect match, the final percent identity is 90%. In another example, a 90 base subject sequence is compared to a 100 base query sequence. In this case, the deletion is an internal deletion, so that no base is present 5 'or 3' of the subject sequence that does not match / align with the query. In this case, FASTD
The percent identity calculated by B is not manually corrected. Again, only the 5 ′ or 3 ′ base of the subject sequence that does not match / align with the query sequence is manually corrected. No other manual correction is made for the purposes of the present invention.

For example, a polypeptide having an amino acid sequence that is at least 95% “identical” to the query amino acid sequence of the present invention means that the target polypeptide sequence has 100 target amino acid sequences each of the query amino acid sequence. It is intended to be identical to the query sequence except that it may contain up to 5 amino acid changes per amino acid. In other words, at least 95 in the query amino acid sequence
To obtain a polypeptide having an amino acid sequence that is% identical, up to 5% of the amino acid residues in the subject sequence can be inserted, deleted, (indels) or replaced with another amino acid. These modifications of the reference sequence can occur at the amino-terminal or carboxy-terminal position of the reference amino acid sequence, or can occur anywhere between those terminal positions, individually between residues in the reference sequence, or the reference sequence Interspersed in any one or more of the consecutive groups.

In practice, any particular polypeptide may, for example, against the amino acid sequence shown in Table 1 (SEQ ID NO: Y) or against the amino acid sequence encoded by the cDNA contained in the deposited clone. At least 80%
, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity can conventionally be determined using known computer programs. The best overall match between the query sequence (the sequence of the invention) and the subject sequence (
The preferred method for determining (also referred to as global sequence alignment) is Bru
tag et al. (Comp. App. Biosci. 6: 237-245 (1990).
)) Can be determined using the FASTDB computer program. In sequence alignment, the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The above overall sequence alignment results are given in percent identity. FASTD
The preferred parameters used for the B amino acid alignment are: Matrix = PAM
0, k-tuple = 2, Mismatch Penalty = 1, Joni
ng Penalty = 20, Randomization Group Le
ngth = 0, Cutoff Score = 1, Window Size =
Sequence length, Gap Penalty = 5, Gap Size Penalty
= 0.05, Window Size = 500 or the length of the target amino acid sequence (whichever is shorter).

If the subject sequence is shorter than the query sequence (not because of an internal deletion) due to an N-terminal or C-terminal deletion, a manual correction must be made to the result. This is because the FASTDB program does not consider N-terminal and C-terminal truncations of the subject sequence when calculating the overall percent identity. For subject sequences that are cleaved at the N- and C-termini, the percent identity compared to the query sequence is the percent of the subject sequence that does not match / align with the corresponding subject residue as a percentage of the total bases of the query sequence. It is corrected by calculating the total number of residues in the query sequence that are terminal and C-terminal. Whether a residue is matched / aligned is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated using the specific parameters by the FASTDB program described above to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. N of the target sequence that does not match / align with the query sequence
Only the terminal and C-terminal residues are considered for the purpose of manually adjusting the percent identity score. That is, only the query residues are located outside the farthest N-terminal and C-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. Deletions occur at the N-terminus of the subject sequence and therefore the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. The 10 unpaired residues are 10% of the sequence (non-matching N-terminus and C
Number of residues at the end / total number of residues in the query sequence), resulting in FAST
10% is subtracted from the percent identity score calculated by the DB program. If the remaining 90 residues are a perfect match, the final percent identity is 90%. In another example, a 90 residue subject sequence is compared to a 100 residue query sequence. In this case, the deletion is an internal deletion, so there are no N-terminal or C-terminal residues of the subject sequence that do not match / align with the query sequence. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only those residue positions outside the N- and C-termini of the subject sequence that do not match / align with the query sequence shown in the FASTDB alignment are manually corrected.
No other manual correction is made for the purposes of the present invention.

A variant may include changes in the coding region, non-coding region, or both. Particularly preferred produce silent substitutions, additions or deletions,
Polynucleotide variants that contain changes that do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Furthermore, 5 in any combination
Also preferred are variants in which -10, 1-5, or 1-2 amino acids are substituted, deleted, or added. Polynucleotide variants may be optimized for various reasons (eg, optimization of codon expression for a particular host (e.g., codons in human mRNA, E. coli)).
for a preferred codon by a bacterial host such as E. coli))
Can be generated.

A naturally occurring variant is called an “allelic variant” and refers to one of several alternative forms of a gene that occupies a given locus on the chromosome of an organism (Genes II, Lewin, B., Ed John Wiley &
Sons, New York (1985)). These allelic variants can vary at either the polynucleotide level and / or the polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants can be produced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNA technology, the variant is
It can be made to improve or change the properties of the polypeptides of the invention. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without a substantial loss of biological function. Ron et al. Biol. Ch
em. 268: 2984-2988 (1993), 3, 8, or 2
A variant KGF protein with heparin binding activity was reported even after deletion of the 7 amino-terminal amino acid residues. Similarly, interferon gamma has been deleted after deleting 8-10 amino acid residues from the carboxy terminus of the protein.
Showed higher activity up to 0-fold (Dobeli et al., J. Biotechnol
ogy 7: 199-216 (1988)).

Furthermore, extensive evidence demonstrates that variants often retain activity similar to the biological activity of naturally occurring proteins. For example, Gayle and co-workers (J. Biol. Chem 268: 22105-22111 (1
993)) performed extensive mutational analysis of the human cytokine IL-1a. They used random mutagenesis to create over 3,500 individual IL-1a variants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were tested at all potential amino acid positions. The researchers found that "the majority of the molecules can be altered with little effect on either [binding or biological activity]". (See summary.) In fact, of over 3,500 nucleotide sequences tested,
Only 23 unique amino acid sequences produced proteins that were significantly different in activity from the wild type.

Furthermore, even if the deletion of one or more amino acids from the N-terminus or C-terminus of the polypeptide results in the modification or deletion of one or more biological functions, other biological activities are still present. Can be retained. For example, the ability of a deletion variant to induce and / or bind an antibody that recognizes a secreted form is such that fewer than the majority of the secreted form residues are removed from the N-terminus or C-terminus. Seems to be retained. Whether a particular polypeptide lacking the N-terminal or C-terminal residue of a protein retains such immunogenic activity is determined by conventional methods described herein, and so on. Otherwise, it can be easily determined by conventional methods known in the art.

Accordingly, the present invention further includes polypeptide variants that exhibit substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to common rules known in the art so as to have little effect on activity. For example, guidance on how to make phenotypically silent amino acid substitutions can be found in Bowie et al., Science 247: 1.
306-1310 (1990), where the authors point out that there are two main strategies for studying the tolerance of amino acid sequences to changes.

The first strategy takes advantage of the tolerance of amino acid substitutions by natural selection during the course of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids appear to be important for protein function. In contrast, amino acid positions whose substitutions have been tolerated by natural selection indicate that these positions are not critical to protein function. Thus, positions that tolerate amino acid substitutions can be altered, but still maintain the biological activity of the protein.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions in the cloned gene to identify regions important for protein function. For example, site-directed mutagenesis or alanine scanning mutagenesis (
Introduction of a single alanine mutation at any residue in the molecule) can be used. (
Cunningham and Wells, Science 244: 1081-
1085 (1989)). The resulting mutant molecules can then be tested for biological activity.

As the authors mention, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes appear to be tolerated at specific amino acid positions in the protein. For example, amino acid residues that are almost buried (within the tertiary structure of the protein) require nonpolar side chains, but the characteristics of surface side chains are generally rarely conserved. In addition, tolerated conservative amino acid substitutions include substitution of aliphatic or hydrophobic amino acids Ala, Val, Leu, and Ile; substitution of hydroxyl residues Ser and Thr; substitution of acidic residues Asp and Glu; Asn of amide residue
And Gln substitutions, substitution of basic residues Lys, Arg, and His; substitution of aromatic residues Phe, Tyr, and Trp, and small size amino acids Ala, Ser, Thr, Met, and Gly Includes substitution.

In addition to conservative amino acid substitutions, the variants of the invention include (i) substitution with one or more non-conservative amino acid residues, wherein the substituted amino acid residues are encoded by the genetic code An amino acid residue may or may not be, or (ii) a substitution with one or more amino acid residues having a substituent, or (i
ii) fusion of the mature polypeptide with another compound (eg, a compound (eg, polyethylene glycol) to increase the stability and / or solubility of the polypeptide), or (iv) additional amino acids (eg, IgG Fusion of a polypeptide with an Fc fusion region peptide, or a leader or secretory sequence, or a sequence that facilitates purification, or (v) another compound (eg, but not limited to, albumin) Modified albumin (eg, US Pat. No. 5,876,969 issued March 2, 1999, EP 0 413 622, and US Pat. No. 5,766, issued June 16, 1998). 883 (
These include fusions with)))), which is incorporated herein by reference in its entirety. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

For example, polypeptide variants that include amino acid substitutions of charged amino acids with other charged amino acids or neutral amino acids can produce proteins with improved properties (eg, less aggregation) . Aggregation of pharmaceutical formulations results in both decreased activity and increased clearance due to the aggregate's immunogenic activity. (Pinkcard et al., Clin. Exp. Immunol. 2: 331
-340 (1967); Robbins et al., Diabetes 36: 838-
845 (1987); Cleland et al., Crit. Rev. Therapeu
tic Drug Carrier Systems 10: 307-377 (
1993)).

Further embodiments of the invention include at least one amino acid substitution,
Amino acids of the invention having an amino acid sequence that does not exceed amino acid substitutions, even more preferably does not exceed 40 amino acid substitutions, even more preferably does not exceed 30 amino acid substitutions, and even more preferably does not exceed 20 amino acid substitutions It relates to a polypeptide comprising a sequence. Of course, in order of increasing preference, the peptide or polypeptide contains at least one amino acid substitution, but 10, 9, 8,
It is highly preferred to have an amino acid sequence comprising an amino acid sequence of the invention that does not exceed 7, 6, 5, 4, 3, 2, or 1 amino acid substitution. In certain embodiments, the number of additions, substitutions, and / or deletions in the amino acid sequences of the invention or fragments thereof (eg, mature forms and / or other fragments described herein) are 1-5, 5-10, 5-25, 5-50, 10-50, or 50
~ 150, with conservative amino acid substitutions being preferred.

(Polynucleotide and polypeptide fragments)
The invention also relates to polynucleotide fragments of the polynucleotides of the invention.

In the present invention, a “polynucleotide fragment” refers to a short polynucleotide having the following nucleic acid sequence: coding for a polypeptide contained in the deposited clone or encoded by the cDNA in the deposited clone. Part of the nucleic acid sequence shown in SEQ ID NO: X or the complementary strand thereof, or part of the polynucleotide sequence encoding the polypeptide of SEQ ID NO: Y. The nucleotide fragments of the present invention preferably have at least about 15 nt, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about The length is 150 nt. For example, a fragment of “at least about 20 nt in length” is intended to include 20 or more contiguous bases derived from the cDNA sequence contained in the deposited clone or the nucleotide sequence shown in SEQ ID NO: X.
In this context, “about” is approximately the stated value (at either or both ends, by a few (5, 4, 3, 2, or 1) more or less nucleotides. Value). These nucleotide fragments have uses including, but not limited to, use as diagnostic probes and primers, as discussed herein. Of course, larger fragments (eg 50, 150, 500, 600, 2000 nucleotides)
Is preferred.

Furthermore, as a representative example of the polynucleotide fragment of the present invention, for example,
Examples include fragments comprising or consisting of the following approximate nucleotide numbers: 1-50, 51-100, 101-150, 151-20.
0, 201-250, 251-300, 301-350, 351-400, 40
1-450, 451-500, 501-550, 551-600, 651-70
0, 701-750, 751-800, 800-850, 851-900, 90
1-950, 951-1000, 1001-1050, 1051-1100, 1
101 to 1150, 1151 to 1200, 1201 to 1250, 1251 to 13
00, 1301-1350, 1351-1400, 1401-1450, 145
1-1500, 1501-1550, 551-1600, 1601-1650
1651 to 1700, 1701 to 1750, 1751 to 1800, 1801
1850, 1851 to 1900, 1901 to 1950, 1951 to 2000, or 2001 to the end of SEQ ID NO: X, their complementary strand, or cDNA contained in the deposited clone. In this context, "about" is
Specifically stated ranges, and ranges at either or both ends, include ranges that are larger or smaller by several (5, 4, 3, 2, or 1) nucleotides. Preferably, these fragments encode polypeptides having biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides that hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also included in the invention, as well as polypeptides encoded by these polynucleotides.

In the present invention, the “polypeptide fragment” refers to an amino acid sequence that is part of the amino acid sequence contained in SEQ ID NO: Y or encoded by the cDNA contained in the deposited clone. A protein (polypeptide) fragment can be “free-standing” or within a larger polypeptide (the fragment is part or region (most preferably as a single contiguous region)) Forming). Representative examples of the polypeptide fragments of the present invention include, for example, fragments comprising the following approximate amino acid numbers or consisting of the following approximate amino acid numbers: 1-20, 21-40, 41- 60, 61-80, 81-100, 10
2 to 120, 121 to 140, 141 to 160, or 161 to the end of the coding region. In addition, polypeptide fragments can be about 20, 30, 40, 50,
60, 70, 80, 90, 100, 110, 120, 130, 140, or 1
Can be 50 amino acids long. In this context, “about” means
Specifically included ranges or values, and ranges or values that are larger or smaller by several (5, 4, 3, 2, or 1) amino acids at either or both ends. Polynucleotides encoding these polypeptides are also included in the present invention.

Preferred polypeptide fragments include secreted proteins and mature forms. Further preferred polypeptide fragments include secreted proteins or mature forms having a contiguous series of deleted residues from the amino terminus or carboxy terminus or both. For example, any number of amino acids (range 1-60) can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids (range 1-30) can be
It can be deleted from the carboxy terminus of the secreted protein or mature form. further,
Any combination of the above amino and carboxy terminal deletions is preferred.
Likewise preferred are polynucleotides encoding these polypeptide fragments.

Polypeptides and polynucleotide fragments characterized by structural or functional domains (e.g., α-helix and α-helix forming regions, β-sheets and β-sheet forming regions, turns and turn forming regions,
Coils and coil-forming regions, hydrophilic regions, hydrophobic regions, α amphiphilic regions, β amphiphilic regions, flexible regions, surface forming regions, substrate binding regions, and fragments containing high antigenic index regions) Also preferred. The polypeptide fragment of SEQ ID NO: Y contained within the conserved domain is specifically contemplated by the present invention. In addition, polynucleotides encoding these domains are also contemplated.

Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those that exhibit similar activity but are not necessarily identical to the activity of the polypeptides of the invention. The biological activity of this fragment can include an improved desired activity or a reduced undesirable activity. Polynucleotides encoding these polypeptide fragments are also
It is included in the present invention.

Preferably, the polynucleotide fragment of the present invention encodes a polypeptide that exhibits functional activity. A polypeptide that exhibits “functional activity” refers to a polypeptide that can exhibit one or more known functional activities associated with the full-length (complete) polypeptide of the protein of the invention. Such functional activities include biological activity, antigenicity [ability to bind (or compete with, bind to) an antibody against the polypeptide of the present invention], immunogenicity ( Ability to generate antibodies that bind to the polypeptides of the invention), the ability to form multimers with the polypeptides of the invention, and the ability to bind to receptors or ligands for the polypeptides of the invention. It is not limited.

The functional activity of the polypeptides of the invention, and fragments, variants, derivatives, and analogs thereof can be assayed by a variety of methods.

For example, in one embodiment assaying for the ability of a polypeptide of the invention to bind to or compete with a full-length polypeptide of the invention for binding to an antibody, various immunoassays known in the art may be used. Can be. Such assays include radioimmunoassays, ELISAs (enzyme-linked immunosorbent assays), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (eg, colloidal gold, enzymes or Radioisotope labeling), Western blot, precipitation reaction, aggregation assay (eg, gel aggregation assay, hemagglutination assay), complement binding assay,
Competitive and non-competitive assay systems that use techniques such as immunofluorescence assays, protein A assays, and immunoelectrophoresis assays include, but are not limited to. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detection of binding in an immunoassay and are within the scope of the present invention.

In another embodiment, when the ability of a ligand for an identified polypeptide of the present invention, or a polypeptide fragment, variant, or derivative of the present invention to multimerize is assessed, binding may be reduced, eg, It can be assayed by means well known in the art such as non-reducing gel chromatography, protein affinity chromatography, and affinity blotting.
See generally Physicky, E .; Et al., 1995, Microbiol. Rev
. 59: 94-123. In another embodiment, the physiological correlation (signal transduction) of binding of a polypeptide of the invention to its substrate can be assayed.

In addition, the assays described herein (see Examples) and other assays known in the art include polypeptides of the present invention and fragments, variants, derivatives, and analogs of the present invention. Can be routinely applied to measure the ability to elicit relevant biological activity (either in vitro or in vivo) related to the biological activity of the polypeptide. Other methods are known to those skilled in the art and are within the scope of the present invention.

(Epitope and antibody)
The invention relates to a polypeptide encoded by an epitope of a polypeptide having the amino acid sequence of SEQ ID NO: Y, or a polynucleotide that is hybridized to a polynucleotide sequence contained in ATCC Deposit No. Z, or to the complement of the sequence of SEQ ID NO: X. An epitope of a peptide sequence, or an epitope of a polypeptide sequence encoded by a polynucleotide sequence contained in ATCC Deposit No. Z under stringent hybridization conditions or lower stringency hybridization conditions as defined above Or a polypeptide comprising them. The present invention further includes a polynucleotide sequence encoding an epitope of a polypeptide sequence of the present invention (eg, the sequence disclosed in SEQ ID NO: X), a polynucleotide sequence of a complementary strand of a polynucleotide sequence encoding the epitope of the present invention, And a polynucleotide sequence that hybridizes to a complementary strand under stringent hybridization conditions as defined above or under lower stringency hybridization conditions.

The term “epitope” as used herein refers to a portion of a polypeptide having antigenic or immunogenic activity in an animal, preferably in a mammal, and most preferably in a human. In a preferred embodiment,
The present invention includes a polypeptide comprising an epitope and a polynucleotide encoding this polypeptide. An “immunogenic epitope” as used herein is as determined by any method known in the art (eg, by the methods for producing antibodies described below). , Defined as part of a protein that elicits an antibody response in an animal (eg, Geysen et al., Proc
. Natl. Acad. Sci. USA 81: 3998-4002 (1983
)checking). The term “antigenic epitope” as used herein refers to an antibody as determined by any method well known in the art (eg, by the immunoassay described herein). Is defined as the part of a protein that can immunospecifically bind to its antigen. Immunospecific binding excludes non-specific binding but does not need to exclude cross-reactivity with other antigens. Antigenic epitopes need not be immunogenic.

Fragments that function as epitopes can be produced by any conventional method. (For example, Houghten, Proc. Natl. Acad. Sci.
. USA 82: 5131-5135 (1985). This is further described in US Pat. No. 4,631,211).

In the present invention, the antigenic epitope preferably comprises at least 4, at least 5, at least 6, at least 7 amino acid sequences, more preferably at least 8, at least 9, at least 10, at least 11, at least 12.
At least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50 amino acid sequences, and most preferably between about 15 and about 30 amino acids. Preferred polypeptides containing immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55
, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acid residues in length. Further non-exclusively preferred antigenic epitopes include the antigenic epitopes disclosed herein and portions thereof. Antigenic epitopes are useful (eg, to elicit antibodies (including monoclonal antibodies) that specifically bind to the epitope). Preferred antigenic epitopes include the antigenic epitopes disclosed herein and any combination of 2, 3, 4, 5 or more of these antigenic epitopes. Antigenic epitopes can be used as target molecules in immunoassays. (For example, Wilson et al., Cell 37: 767-7.
78 (1984); Sutcliffe et al., Science 219: 660-
666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (For example, Sutcliffe et al. (Supra); Wils
on et al. (supra); Chow et al., Proc. Natl. Acad. Sci. USA
82: 910-914; and Bittle et al., J. MoI. Gen. Virol. 6
6: 2347-2354 (1985)). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. A polypeptide comprising one or more immunogenic epitopes can be presented to elicit an antibody response against an animal system (eg, rabbit or mouse) with a carrier protein (eg, albumin) or the polypeptide is If long enough (at least about 25 amino acids), the polypeptide can be presented without a carrier. However, immunogenic epitopes containing as few as 8-10 amino acids
It has been shown to be sufficient to elicit antibodies that can (at least) bind to linear epitopes of the denatured polypeptide (eg, in Western blotting).

The epitope-bearing polypeptides of the invention can be used to induce antibodies according to methods well known in the art. This method includes, but is not limited to, in vivo immunization, in vitro immunization, and phage display methods.
For example, Sutcliffe et al., Supra; Wilson et al., Supra; and Bitt.
le et al. Gen. Virol. 66: 2347-2354 (1985). When using in vivo immunization, animals can be immunized with free peptide; however, anti-peptide antibody titers can be achieved with a peptide on a macromolecular carrier (eg, keyhole limpet hemocyanin (KLH) or tetanus toxoid). Can be boosted by combining. For example, peptides containing cysteine residues can be attached to the carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). On the other hand, other peptides can be bound to the carrier using more common binding agents such as glutaraldehyde. Animals such as rabbits, rats, and mice can use either free peptides or carrier-binding peptides, for example, emulsions (about 100 μg of peptide or carrier protein and Freund's adjuvant or any known to stimulate an immune response. It is immunized by intraperitoneal injection and / or intradermal injection (including other adjuvants). Several booster injections may be required, for example, at intervals of about 2 weeks, to provide useful titers of anti-peptide antibodies. This titer can be detected, for example, by an ELISA assay using free peptide adsorbed on a solid surface. The titer of anti-peptide antibodies in serum from immunized animals is increased by selection of anti-peptide antibodies (eg, by adsorption of peptides on solid supports and elution of selected antibodies according to methods well known in the art) Can do.

As will be appreciated by those skilled in the art and as discussed above, the polypeptides of the invention comprising immunogenic or antigenic epitopes can be fused to other polypeptide sequences. For example, the polypeptide of the present invention may be an immunoglobulin (Ig
A, IgE, IgG, IgM) constant domains or parts thereof (CH1, C
H2, CH3, or any combination thereof and portions thereof) or albumin (recombinant albumin (eg, US Pat. No. 5,876,969 issued March 2, 1999, EP 0 413 622). And U.S. Pat. No. 5,766,883, issued June 16, 1998, which are hereby incorporated by reference in their entirety), but are not limited to) Can be fused to yield a chimeric polypeptide. Such fusion proteins can facilitate purification and increase half-life in vivo. this is,
A chimeric protein consisting of the first two domains of a human CD4-polypeptide and the various domains of the constant region of a mammalian immunoglobulin heavy or light chain is shown. See, for example, EP 394,827; Traunecker et al., Nature, 331: 84-86 (1988). Enhanced delivery of antigens across the epithelial barrier to the immune system has been demonstrated for antigens (eg, insulin) bound to FcRn binding partners such as IgG or Fc fragments (eg, PCT Publication WO 96/22024 and WO99 / 0
4813). IgG fusion proteins with disulfide-linked dimeric structures resulting from disulfide bonds in the IgG moiety are also more effective at binding and neutralizing other molecules than monomeric polypeptides or their fragments alone Was found. For example, Fountoulakis et al. B
iochem. 270: 3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with the gene of interest as an epitope tag (eg, a hemagglutinin (“HA”) tag or flag tag) to detect and purify the expressed polypeptide. Can assist. For example, Ja
The system described by nknecht et al. allows easy purification of non-denaturing fusion proteins expressed in human cell lines (Janknecht et al., 1991,
Proc. Natl. Acad. Sci. USA 88: 8972-897).
In this system, the gene of interest is subcloned into a vaccinia recombinant plasmid so that the open reading frame of this gene is fused at translation to an amino-terminal tag consisting of six histidine residues. This tag serves as a substrate binding domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2 + nitriloacetic acid-agarose columns and histidine-tagged proteins can be selectively eluted using imidazole-containing buffers.

Additional fusion proteins of the invention can be generated through the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as “DNA shuffling”). D
NA shuffling can be utilized to modulate the activity of the polypeptides of the invention, and by using this method, polypeptides with altered activity, as well as agonists and antagonists of these polypeptides, can be generated. In general, U.S. Pat. Nos. 5,605,793; 5,811,238;
No. 0,721; No. 5,834,252; and No. 5,837,458,
As well as Patten et al., Curr. Opinion Biotechnol.
8: 724-33 (1997); Harayama, Trends Biote
chnol. 16 (2): 76-82 (1998); Hansson et al., J. Biol. M
ol. Biol. 287: 265-76 (1999); and Lorenzo
And Blasco, Biotechniques 24 (2): 308-13.
(1998) (each of these patents and publications incorporated herein by reference in its entirety). In one embodiment, modification of the polynucleotides corresponding to SEQ ID NO: X and the polypeptides encoded by these polynucleotides can be accomplished by DNA shuffling. D
NA shuffling involves assembling two or more DNA segments to produce changes in a polynucleotide sequence by homologous recombination or site-specific recombination. In another embodiment, the polynucleotide of the invention, or the encoded polypeptide thereof, is error-prone prior to recombination.
ne) Can be modified by subjecting it to random mutagenesis by PCR, random nucleotide insertion or other methods. In another embodiment, one or more components, motifs, sections, portions, domains, fragments, etc. of a polynucleotide encoding a polypeptide of the invention are one or more components, motifs of one or more heterologous molecules. , Sections, parts, domains, fragments and the like.

(antibody)
Further, a polypeptide of the invention can be a polypeptide, polypeptide fragment, or SEQ ID NO: Y of the invention (as determined by immunoassays well known in the art for assaying specific antibody antigen binding). Variants, and / or
Or an antibody that immunospecifically binds to an epitope and a T cell antigen receptor (T
CR). The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies,
Multispecific antibodies, human antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ′) fragments, fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies (eg, Including, but not limited to, anti-Id antibodies to the antibodies of the invention), and any of the epitope-binding fragments described above. As used herein, the term “antibody” refers to
Immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that immunospecifically binds to an antigen. The immunoglobulin molecules of the invention can be any type of immunoglobulin molecule (eg, IgG, IgE, I
gM, IgD, IgA, and IgY), any class (eg, IgG1, I
gG2, IgG3, IgG4, IgA1 and IgA2) or any subclass. In a preferred embodiment, the immunoglobulin molecule of the present invention is IgG
1. In another preferred embodiment, the immunoglobulin of the present invention is IgG4.

Most preferably, the antibody is a human antigen binding antibody fragment of the invention, which includes Fab, Fab ′ and F (ab ′) 2, Fd, single chain Fvs (sc
Fv), single chain antibodies, disulfide bond Fvs (sdFv) and VL or V
Examples include, but are not limited to, fragments containing any of the H domains. An antigen-binding antibody fragment comprising a single chain antibody may comprise the variable region alone or in combination with all or part of the following: hinge region, CH1 domain, C
H2 domain and CH3 domain. Also included in the invention are antigen-binding fragments that also include any combination of variable region and hinge region, CH1 domain, CH2 domain, and CH3 domain. The antibodies of the invention can be derived from any animal origin including birds and mammals. Preferably, the antibody is human, murine (eg, mouse and rat), donkey, ship rabbit (
ship rabbit), goat, guinea pig, camel, horse, or chicken. As used herein, a “human” antibody includes an antibody having the amino acid sequence of a human immunoglobulin and is transgenic for a human immunoglobulin library or one or more human immunoglobulins and is endogenous. Antibodies isolated from animals that do not express sex immunoglobulin (as described below and as described, for example, in US Pat. No. 5,939,598 by Kucherlapati et al.).

The antibodies of the invention can be monospecific, bispecific, trispecific or more multispecific antibodies. Multispecific antibodies can be specific for different epitopes of a polypeptide of the invention, or both a polypeptide of the invention and a heterologous epitope (eg, a heterologous polypeptide or a solid support material). Specific. For example, PCT publication WO 93/17715;
2/08802; WO 91/00360; WO 92/05793; Tu
tt et al. Immunol. 147: 60-69 (1991); U.S. Pat.
, 474,893, 4,714,681, 4,925,648,
5,573,920, 5,601,819; Kostelny et al.,
J. et al. Immunol. 148: 1547-1553 (1992).

The antibodies of the invention can be described or specified with respect to an epitope or portion of a polypeptide of the invention to which they recognize or specifically bind. This epitope or portion of the polypeptide is specified, for example, by the N-terminal and C-terminal positions, as described herein by size in consecutive amino acid residues, or as listed in the tables and figures Can be Antibodies that specifically bind to any epitope or polypeptide of the invention can also be eliminated. Accordingly, the present invention includes antibodies that specifically bind a polypeptide of the present invention and allow for elimination of the polypeptide of the present invention.

The antibodies of the invention can also be described or specified for their cross-reactivity. Antibodies that do not bind any other analog, ortholog or homolog of a polypeptide of the invention are included. At least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% and at least 50% identity to the polypeptides of the invention Antibodies that bind a polypeptide having (as calculated using methods known in the art and methods described herein) are also included in the invention. In certain embodiments, the antibodies of the present invention cross-react with mouse homologues, rat homologues and / or rabbit homologues of human proteins and corresponding epitopes thereof. 9 for polypeptides of the invention
<5%, <90%, <85%, <80%, <75%, <70%, 6
Bind polypeptides having less than 5%, less than 60%, less than 55% and less than 50% identity (as calculated using methods known in the art and methods described herein) Non-antibodies are also included in the invention. In certain embodiments, the cross-reactivity is any monospecific antigenic or immunogenic polypeptide disclosed herein, or 2, 3, 4, 5 or more specific It relates to a combination of antigenic and / or immunogenic polypeptides. Further included in the present invention are antibodies that bind a polypeptide encoded by a polynucleotide that hybridizes to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). . The antibody of the present invention also comprises
Their binding affinity for the polypeptides of the invention can be described or specified. Preferred binding affinities are less than 5 × 10 ⁻² M, less than 10 ⁻² M, 5 ×
Less than 10 ⁻³ M, less than 10 ⁻³ M, less than 5 × 10 ⁻⁴ M, less than 10 ⁻⁴ M, and 5 × 10 ⁻⁵ M
Less than 10 ⁻⁵ M, less than 5 × 10 ⁻⁶ M, less than 10 ⁻⁶ M, less than 5 × 10 ⁻⁷ M,
0 less than ^-7 M, 5 × 10 ^-8 less than M, 10 ^-8 less than M, 5 × 10 ^-9 less M, less than 10 ^-9 M, less than 5 × 10 ^-10 M, less than 10 ^-10 M, 5 × Less than 10 ^-11 M, less than 10 ^-11 M,
Less than 5 × 10 ⁻¹² M, less than 10 ⁻¹² M, less than 5 × 10 ⁻¹³ M, less than 10 ⁻¹³ M, 5 ×
Affinities having a dissociation constant or Kd of less than 10 ⁻¹⁴ M, less than 10 ⁻¹⁴ M, less than 5 × 10 ⁻¹⁵ M or less than 10 ⁻¹⁵ M are mentioned.

The invention also provides for binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding (eg, an immunoassay described herein). Antibodies that competitively inhibit are provided. In preferred embodiments, the antibody is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%, competitively binding to the epitope. Inhibit.

The antibodies of the present invention can act as agonists or antagonists of the polypeptides of the present invention. For example, the invention includes antibodies that disrupt either partially or completely receptor / ligand interactions with the polypeptides of the invention. Preferably, an antibody of the invention binds an antigenic epitope disclosed herein, or a portion thereof. The present invention has features of both receptor-specific antibodies and ligand-specific antibodies. The present invention also features receptor-specific antibodies that do not interfere with ligand binding but interfere with receptor activation. Receptor activation (ie, signal transduction) can be determined by techniques described herein, otherwise techniques known in the art. For example, receptor activation can be determined by detecting its substrate by receptor phosphorylation (eg, tyrosine or serine / threonine), or immunoprecipitation followed by Western blot analysis (eg, as described above). . In certain embodiments, in the absence of the antibody, the ligand activity or receptor activity is at least 95% of that activity,
At least 90%, at least 85%, at least 80%, at least 75%,
Antibodies that inhibit at least 70%, at least 60%, or at least 50% are provided.

The present invention also recognizes receptor-specific antibodies that interfere with both ligand binding and receptor activation, and receptor-ligand complexes, and preferably does not specifically recognize unbound receptors or unbound ligands. It has the characteristics of a receptor-specific antibody. Similarly, the present invention binds ligands and neutralizes antibodies that prevent ligand binding to the receptor, and ligands, thereby preventing receptor activation, but does not prevent the ligand from binding the receptor. Contains antibodies. Furthermore, the present invention includes antibodies that activate the receptor. These antibodies can act as receptor agonists, i.e., enhance or activate either all or a subset of the biological activation of ligand-mediated receptor activation, e.g. by inducing receptor dimerization. obtain. This antibody can be specified as an agonist, antagonist or inverse agonist for biological activity, including specific biological activity of the peptides of the invention disclosed herein. The antibody agonist can be produced using a method known in the art. See, for example, PCT Publication WO 96/40281; US Pat. No. 5,811,097; Deng et al., Blood 92 (6): 1981-19.
88 (1998); Chen et al., Cancer Res. 58 (16): 366
8-3678 (1998); Harrop et al., J. Biol. Immunol. 161 (4
): 1786-1794 (1998); Zhu et al., Cancer Res: 58
(15): 3209-3214 (1998); Immunol
. 160 (7): 3170-3179 (1998); Prat et al., J. Biol. Cell
. Sci. 111 (Pt2): 237-247 (1998); Pitald et al.,
J. et al. Immunol. Methods 205 (2): 177-190 (199
7); Liautard et al., Cytokine 9 (4): 233-241 (1)
997); Carlson et al., J. MoI. Biol. Chem. 272 (17): 11
295-11301 (1997); Taryman et al., Neuron 14 (4
): 755-762 (1995); Muller et al., Structure 6 (
9): 1153-1167 (1998); Bartunek et al., Cytokin
e 8 (1): 14-20 (1996) (all of the above references are incorporated herein by reference in their entirety).

The antibodies of the invention can be used, for example, but not limited to, to purify, detect, and target the polypeptides of the invention. These include both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibody has use in an immunoassay to qualitatively and quantitatively measure the level of a polypeptide of the invention in a biological sample. For example, Harl
ow et al., Antibodies: A Laboratory Manual, (
Cold Spring Harbor Laboratory Press,
2nd edition, 1988) (incorporated herein by reference in its entirety).

As discussed in further detail below, the antibodies of the present invention can be used either alone or in combination with other compounds. The antibody can further be recombinantly fused to the heterologous polypeptide at the N-terminus or C-terminus, or chemically conjugated (including covalent and non-covalent) to the polypeptide or other composition. . For example, the antibodies of the invention can be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. For example, PCT publication WO92 / 084
95; WO 91/14438; WO 89/12624; US Pat. No. 5,314
995; and European Patent No. 396,387.

The antibodies of the present invention can be modified (ie, covalent attachment
attachment) includes derivatives) by covalent attachment of any type of molecule to the antibody such that it does not prevent the antibody from producing an anti-idiotypic response. For example, without limitation, antibody derivatives include, for example, glycosylation, acetylation, pegylation, phosphorylation (phosphophylatio).
n), amidation, known protecting / blocking groups
And antibodies modified by derivatization with, proteolytic cleavage, linking to cellular ligands or other proteins. A number of optional chemical modifications can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more non-classical amino acids.

The antibodies of the present invention can be produced by any suitable method known in the art.
Polyclonal antibodies against the antigen of interest can be produced by various procedures well known in the art. For example, the polypeptides of the present invention can be administered to a variety of host animals (including but not limited to rabbits, mice, rats, etc.) to induce the production of serum containing polyclonal antibodies specific for the antigen. . Various adjuvants can be used to increase the immunological response, depending on the host species, and mineral gels such as Freund (complete and incomplete), aluminum hydroxide (
mineral gel), surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentials such as BCG (Bacille Calmette-Guerin) and corynebacterium parvum Including, but not limited to, human adjuvants useful. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or combinations thereof. For example, monoclonal antibodies can be produced using the following hybridoma techniques known in the art, eg, Har
low et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press
, 2nd edition, 1988); Hammerling et al., Monoclonal An.
tibodies and T-Cell Hybridomas 563-6
81 (Elsevier, NY 1981) (the above references are incorporated by reference in their entirety). As used herein, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples (eg, Example 16). In a non-limiting example, mice can be immunized with a polypeptide of the invention or cells that express such a peptide. Once an immune response is detected (eg, an antibody specific for the antigen is detected in the serum of the mouse)
The mouse spleen is collected and splenocytes are isolated. The splenocytes are then fused to any suitable myeloma cells (eg, cells from cell line SP20 available from ATCC) by well-known techniques. Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed by methods known in the art for cells secreting antibodies capable of binding to the polypeptides of the invention. Ascites fluid, which generally contains high levels of antibodies
) Can be produced by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides a method for producing an antibody produced by a method comprising culturing a monoclonal antibody and a hybridoma cell secreting the antibody of the present invention, wherein preferably the hybridoma comprises: For hybridoma clones that fuse myeloma cells and spleen cells isolated from a mouse immunized with an antigen of the invention and then secrete an antibody capable of binding to the polypeptide of the invention,
Produced by screening for hybridomas arising from the fusion.

Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, the Fab and F (ab ′) 2 fragments of the present invention are (Fa
It can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce b fragments) or pepsin (to produce F (ab ′) 2 fragments). The F (ab ′) 2 fragment contains the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For example, the antibodies of the present invention can also be produced using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences that encode them. In certain embodiments, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (eg, human or mouse). Phage expressing an antigen binding domain that binds to the antigen of interest can be selected or identified using the antigen (eg, using a labeled antibody or antigen bound or captured to a solid surface or bead). Phage used in these methods are typically phage having Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either phage gene III protein or phage gene VIII protein. Filamentous phage containing fd and M13 binding domains expressed from.
Examples of phage display methods that can be used to make the antibodies of the invention include the methods disclosed below: Brinkman et al. Immu
nol. Methods 182: 41-50 (1995); Ames et al., J. Biol.
Immunol. Methods 184: 177-186 (1995); Ke
castleborough et al., Eur. J. et al. Immunol. 24: 952-95
8 (1994); Persic et al., Gene 187 9-18 (1997);
Burton et al., Advances in Immunology 57:19
1-280 (1994); PCT application number PCT / GB91 / 01134; PC
T Publication WO 90/02809; WO 91/10737; WO 92/010
47; WO 92/18619; WO 93/11236; WO 95/159
82; WO 95/20401; and US Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717.
No. 5,427,908 No. 5,750,753 No. 5,821, No.
No. 047; No. 5,571,698; No. 5,427,908; No. 5,5
No. 16,637; No. 5,780,225; No. 5,658,727; No. 5,733,743 and No. 5,969,108 (each of which is herein incorporated by reference) Which is incorporated by reference in its entirety).

As described in the above references, after phage selection, the region encoding the antibody derived from the phage is isolated to produce the entire antibody, including human antibodies, or any other desired antigen-binding fragment and And can be expressed in any desired host, including, for example, mammalian cells, insect cells, plant cells, yeast and bacteria, as described in detail below. For example, techniques for recombinantly producing Fab, Fab ′ and F (ab ′) 2 fragments can also be used using methods known in the art, and such methods are disclosed below. : PC
T Publication WO 92/22324; Mullinax et al., BioTechnique
es 12 (6): 864-869 (1992); and Sawai et al., AJR.
I 34: 26-34 (1995); and Better et al., Science.
240: 1041-1043 (1998) (these references are incorporated by reference in their entirety).

Examples of techniques that can be used to produce single chain Fvs and antibodies include US Pat. Nos. 4,946,778 and 5,258,498; Huston
Et al., Methods in Enzymology 203: 46-88 (19
91); Shu et al., PNAS 90: 7995-7999 (1993); and Skerra et al., Science 240: 1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, the use of chimeric, humanized or human antibodies may be preferred. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species (eg, antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region). Methods for producing chimeric antibodies are known in the art. For example, Morris
on, Science 229: 1202 (1985); Oi et al., BioTec.
hniques 4: 214 (1986); Gillies et al. (1989) J
. Immunol. Methods 125: 191-202; US Pat. No. 5,
807,715; 4,816,567; and 4,816,397, which are hereby incorporated by reference in their entirety.
. A humanized antibody is an antibody molecule derived from a non-human species antibody that binds to a desired antigen having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule. . Often, framework residues within the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter (preferably improve) antigen binding. These framework substitutions are identified by methods well known in the art, such as modeling CDR and framework residue interactions to identify framework residues important for antigen binding, and at specific positions. By sequence comparison to identify unusual framework residues. (See, eg, Queen et al., US Pat. No. 5,585,0.
89; Riechmann et al., Nature 332: 323 (1988), which are hereby incorporated by reference in their entirety. )
Antibodies can be humanized using a variety of techniques known in the art including, for example: CDR-transplant (European Patent No. 239,400; PCT Publication WO 91/09)
967; U.S. Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (European Patent No. 592,106). No. 519,596; Padlan, Molecular Immunology
28 (4/5): 489-498 (1991); Studnikka et al., P
rotein Engineering 7 (6): 805-814 (1994)
Roguska et al., PNAS 91: 969-973 (1994)), and chain shuffling (US Pat. No. 5).
, 565,332).

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be produced by various methods known in the art, including the phage display method described above using an antibody library derived from human immunoglobulin sequences. U.S. Pat. Nos. 4,444,887, 4,716,111; and PCT Publication W
O 98/46645, WO 98/50433, WO 98/24893, W
See also O 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but are capable of expressing human immunoglobulin genes. For example, a complex of a human heavy chain immunoglobulin gene and a human light chain immunoglobulin gene can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, human variable regions, constant regions and diversity regions (diversity)
region) can be introduced into mouse embryonic stem cells in addition to the human heavy chain gene and human light chain gene. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the normal fashion with a selected antigen (eg, all or part of a polypeptide of the invention). Monoclonal antibodies against the antigen can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgene retained in the transgenic mouse is B
Reconstitute during cell differentiation and then undergo class switching and somatic mutation. Thus, the use of such techniques allows the production of therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar,
Int. Rev. Immunol. 13: 65-93 (1995). For a detailed discussion of this technology for producing human and human monoclonal antibodies and protocols for producing such antibodies, see, eg, PCT Publication WO 98/24893; WO 92/01047; WO 96/340.
96; WO 96/33735; European Patent 0 598 877; US Patent No. 5,
No. 413,923; No. 5,625,126; No. 5,633,425; No. 5,569,825; No. 5,661,016; No. 5,545,806
No. 5,814,318; No. 5,885,793; No. 5,916,
771; and 5,939,598, which are hereby incorporated by reference in their entirety. Further, Abgenix, Inc. (F
companies such as Remont, CA) and Genpharm (San Jose, CA) may be engaged in providing human antibodies against selected antigens using techniques similar to those described above.

Fully humanized antibodies that recognize selected epitopes are referred to as “guided (gui
production using a technique referred to as “ded) selection”. In this approach,
Selected non-human monoclonal antibodies (eg, murine antibodies) are used to guide the selection of fully human antibodies that recognize the same epitope. (Jespers et al., Bio / technology 12: 899-903 (1988)).

Furthermore, antibodies against the polypeptides of the present invention can be utilized in turn to produce anti-idiotypic antibodies that “mimic” the polypeptides of the present invention using techniques well known to those skilled in the art. (For example, Greenspan and Bona, FASEB
J. et al. 7 (5): 437-444; (1989) and Nissinoff, J
. Immunol. 147 (8): 2429-2438 (1991). For example, binding and multimerization of polypeptides.
and / or antibodies that competitively inhibit the binding of a polypeptide of the invention to a ligand are used to produce anti-idiotypes that "mimic" the multimerization and / or binding domains of the polypeptide. And consequently binds to and neutralizes the polypeptide and / or its ligand.
Neutralization of such anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligands. For example, such anti-idiotypic antibodies can be used to bind to a polypeptide of the invention and / or to bind its ligand / receptor, thereby blocking its biological activity.

(Polynucleotide encoding antibody)
The present invention further provides a polynucleotide comprising a nucleotide sequence encoding the antibody and fragments thereof of the present invention. The present invention also relates to an antibody (preferably as defined above) that binds specifically to a polypeptide of the present invention under stringent or lower stringency hybridization conditions, preferably And a polynucleotide that hybridizes to a polynucleotide encoding an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO: Y.

A polynucleotide can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotide is determined. For example, if the nucleotide sequence of an antibody is known, the polynucleotide encoding this antibody can be constructed from chemically synthesized oligonucleotides (eg, Kutmeie).
r et al., BioTechniques 17: 242 (1994)), which briefly includes the following steps: Synthesis of overlapping oligonucleotides containing portions of the sequence encoding this antibody Annealing and ligation of these oligonucleotides, followed by amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody can be generated from nucleic acid from a suitable source. A clone containing a nucleic acid encoding a particular antibody is not available, but if the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or can be synthesized by a suitable source ( For example, an antibody cDNA library, or a cDNA library generated from any tissue or cell that expresses the antibody (eg, a hybridoma cell selected for expression of an antibody of the invention), or a nucleic acid isolated therefrom (Preferably poly A + RNA)), for example, PCR amplification using synthetic primers hybridizable to the 3 ′ and 5 ′ ends of the sequence to identify cDNA clones from a cDNA library encoding the antibody Or use oligonucleotide probes specific for specific gene sequences It may be obtained by cloning. P
The amplified nucleic acid produced by the CR can then be cloned into a replicable cloning vector using any method well known in the art.

Once the antibody nucleotide sequence and the corresponding amino acid sequence are determined,
The nucleotide sequence of an antibody can be determined by methods well known in the art for manipulation of nucleotide sequences (eg, recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (eg,
Sambrook et al., 1990, Molecular Cloning, AL
laboratory Manual, 2nd edition, Cold Spring Har
Bor Laboratory, Cold Spring Harbor, NY
And Ausubel et al., 1998, Current Protocols.
in Molecular Biology, John Wiley & Sons
, NY, refer to the technology described. These are both incorporated herein by reference in their entirety. )) Can be used to generate antibodies having different amino acid sequences, eg, to create amino acid substitutions, deletions, and / or insertions.

In certain embodiments, the amino acid sequences of the heavy and / or light chain variable domains are determined by methods well known in the art (eg, sequence hypervariability) for identification of complementarity determining region (CDR) sequences. Other heavy chains, to determine the region of
And by comparing the variable region of the light chain with a known amino acid sequence). Using conventional recombinant DNA techniques, one or more CDRs can be inserted into the framework region as described above (eg, into the human framework region to humanize non-human antibodies). . This framework region can be naturally occurring or can be a consensus framework region, and preferably can be a human framework region (see, eg, Chothia et al., J. Mol. Biol.278: 457-479 (1
998)). Preferably, the polynucleotide produced by the combination of framework regions and CDRs encodes an antibody that specifically binds to a polypeptide of the invention. Preferably, as discussed above, one or more amino acid substitutions can be made within the framework regions, and preferably the amino acid substitution improves the binding of the antibody to its antigen. Further, such methods produce antibody molecules lacking one or more intrachain disulfide bonds,
It can be used to create amino acid substitutions or deletions of one or more variable region cysteine residues involved in intrachain disulfide bonds. Other changes to the polynucleotide are encompassed by the present invention and in the art.

Furthermore, by splicing a gene from a mouse antibody molecule of appropriate antigen specificity with a gene from a human antibody molecule of appropriate biological activity,
Technology developed to produce "chimeric antibodies" (Morrison et al., Proc
. Natl. Acad. Sci. 81: 851-855 (1984); Neub
erger et al., Nature 312: 604-608 (1984); Take.
da et al., Nature 314: 452-454 (1985)). As described above, a chimeric antibody is a molecule in which different portions are derived from different animal species, and such molecules have variable regions derived from mouse mAb and human immunoglobulin constant regions (eg, humanized antibodies). .

Alternatively, the described techniques for the production of single chain antibodies (US Pat. No. 4,946,46)
778; Bird, Science 242: 423-42 (1988); H
uston et al., Proc. Natl. Acad. Sci. USA 85: 587
9-5883 (1988); and Ward et al., Nature 334: 544.
-54 (1989)) can be adapted for the production of single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. E. Techniques for assembly of functional Fv fragments in E. coli can also be used (Sk
erra et al., Science 242: 1038-1041 (1988)).

(Method of producing antibody)
The antibodies of the present invention can be produced by any method known in the art for synthesizing antibodies, in particular by chemical synthesis, or preferably by recombinant expression techniques.

An antibody of the invention, or a fragment, derivative or analog thereof (eg,
Recombinant expression of the heavy or light chain of the antibody of the present invention or the single chain antibody of the present invention requires the construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding the antibody molecule of the invention or the heavy or light chain of an antibody, or a portion thereof (preferably containing the variable domain of the heavy or light chain) is obtained, production of the antibody molecule Vectors for can be generated by recombinant DNA techniques using techniques well known in the art. Accordingly, methods for preparing a protein by expression of a polynucleotide containing a nucleotide sequence encoding an antibody are described herein. Methods well known to those skilled in the art can be used for the construction of expression vectors containing sequences encoding the antibody and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, the present invention relates to a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. I will provide a.
Such vectors include constant regions of antibody molecules (eg, PCT publication WO86 /
05807; PCT publication WO 89/01036; and US Pat. No. 5,122.
, 464), and the variable domains of the antibodies can be cloned into such vectors for the entire expression of heavy or light chains.

The expression vector is transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibodies of the invention. Accordingly, the present invention includes host cells comprising an antibody of the present invention, or a heavy or light chain thereof, or a polynucleotide encoding a single chain antibody of the present invention operably linked to a heterologous promoter. In a preferred embodiment for the expression of double chain antibodies, vectors encoding both heavy and light chains are co-expressed in a host cell for expression of the entire immunoglobulin molecule, as detailed below. Can be done.

A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such a host expression system represents a vehicle in which the coding sequence of interest can be produced and subsequently purified, but also in situ when transformed or transfected with a sequence encoding the appropriate nucleotide. Represents a cell capable of expressing an antibody molecule of the invention. These include, but are not limited to: bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing sequences encoding the antibody (eg, E. coli, A microorganism such as B. subtilis; a yeast transformed with a recombinant yeast expression vector containing a sequence encoding the antibody (eg, Sacc
insect cell lines infected with recombinant viral expression vectors (eg, baculovirus) containing antibody-encoding sequences; recombinant viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) Or a plant cell line transformed with a recombinant plasmid expression vector (eg, Ti plasmid) containing a sequence encoding an antibody; or a promoter derived from the genome of a mammalian cell (
For example, mammalian cell lines (eg, COS, CHO, BHK) carrying recombinant expression constructs comprising a metallothionein promoter) or a promoter derived from a mammalian virus (eg, adenovirus late promoter; vaccinia virus 7.5K promoter). 293, 3T3 cells). Preferably, Esch
Bacterial cells such as Erichia coli, and more preferably eukaryotic cells, particularly for the expression of whole recombinant antibody molecules, are used for the expression of recombinant antibody molecules. For example, the major immediate early (majo) derived from human cytomegalovirus
Chinese hamster ovary cells (CHO) in combination with vectors such as rintermediate early) gene promoter elements
Mammalian cells such as) are effective expression systems for antibodies (Foecki)
ng et al., Gene 45: 101 (1986); Cockett et al., Bio / T
technology 8: 2 (1990)).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, if large quantities of such proteins are to be produced, vectors directed to high level expression of easily purified fusion protein products may be desired for the production of pharmaceutical compositions of antibody molecules. Such vectors include, but are not limited to, the following: in frame with the region where the antibody-encoding sequence encodes lacZ.
e) can be ligated individually, so that a fusion protein is produced. coli
Expression vector pUR278 (Ruther et al., EMBO J. 2: 1791 (1
983)); pIN vector (Inouye & Inouye, Nucleic)
Acids Res. 13: 3101-3109 (1985); Van Hee
ke & Schuster, J.A. Biol. Chem. 24: 5503-5509
(1989)). pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vector is designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing heterologous genes. This virus grows in Spodoptera frugiperda cells. The sequence encoding the antibody can be individually cloned into a nonessential region of the virus (eg, the polyhedrin gene) and placed under the control of an AcNPV promoter (eg, the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the sequence encoding the antibody of interest may be ligated to an adenovirus transcription / translation control complex, eg, the late promoter and three-part leader sequence. Then
This chimeric gene can be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region (eg, E1 or E3 region) of the viral genome results in a recombinant virus that is viable and capable of expressing antibody molecules in the infected host (eg, Logan & Shenk).
Proc. Natl. Acad. Sci. USA 81: 355-359 (1
984)). A specific initiation signal may also be required for efficient translation of the sequence encoding the inserted antibody. These signals are ATG
Contains the start codon and adjacent sequences. In addition, this initiation codon is the reading frame of the desired coding sequence (to ensure translation of the entire insert (
reading frame) and the phase must be the same. These exogenous translational control signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be increased by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (Bittner et al., Methods in E
nzymol. 153: 51-544 (1987)).

In addition, a host cell line can be chosen, which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
Such modifications (eg glycosylation) and processing of protein products (
For example, cleavage may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. For this purpose, eukaryotic host cells with cellular mechanisms for the precise processing of the first transcription, glycosylation and phosphorylation of the gene product can be used. Such mammalian host cells are CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, WI38, and in particular, for example, BT48
3, including but not limited to breast cancer cell lines such as Hs578T, HTB2, BT20 and T47D, and normal breast cell lines such as CRL7030 and Hs578Bst.

Long-term high-yield production and stable expression of recombinant proteins are preferred. For example, cell lines that stably express antibody molecules can be engineered. Rather than using an expression vector that contains a viral origin of replication, the host cell may use appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.).
And can be transformed with DNA controlled by a selectable marker. Heterogeneous DN
Following the introduction of A, the engineered cells can be grown in enriched media for 1-2 days,
Subsequently, it is switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, and the cell stably integrates the plasmid into its chromosome and proliferates to form a cell growth foci that can in turn be cloned and propagated. It is possible to become a cell line. This method can be advantageously used to engineer cell lines that express antibody molecules. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

A number of selection systems can be used, including herpes simplex virus thymidine kinase (Wigler et al., Cell 11: 223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & S
zybalski, Proc. Natl. Acad. Sci. USA 48: 2
02 (1992)), and adenine phosphoribosyltransferase (L
including but not limited to the genes of owy et al., Cell 22: 817 (1980)), which can be used in tk-, hgprt- or aprt-cells, respectively. Antimetabolite resistance can also be used as a basis for selection of the following genes: dhfr, which confer resistance to methotrexate (W
Igler et al., Natl. Acad. Sci. USA 77: 357 (1980
O'Hare et al., Proc. Natl. Acad. Sci. USA 78:
1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci.
USA 78: 2072 (1981)); neo, which is an aminoglycoside G-
Confer resistance to 418 (Clinical Pharmacy 12: 4
88-505; Wu and Wu, Biotherapy 3: 87-95 (
1991); Tolstoshev, Ann. Rev. Pharmacol. T
oxicol. 32: 573-596 (1993); Mulligan, Sci.
ence 260: 926-932 (1993); and Morgan and
Anderson, Ann. Rev. Biochem. 62: 191-217
(1993); May 1993, TIB TECH 11 (5): 155-21.
5); as well as hygro, which confers resistance to hygromycin (
Santare et al., Gene 30: 147 (1984)). Methods well known in the field of recombinant DNA technology can be routinely applied to select the desired recombinant clone, and such methods are described below: For example, Ausu
bel et al. (eds.), Current Protocols in Molecular.
ar Biology, John Wiley & Sons, NY (1993);
Kriegler, Gene Transfer and Expressio
n, A Laboratory Manual, Stockton Press
, NY (1990); and Chapters 12 and 13, Dracopoli et al. (Eds.), Current Protocols in Human Genetic.
s, John Wiley & Sons, NY (1994);
Garapin et al. Mol. Biol. 150: 1 (1981), which are incorporated herein by reference in their entirety.

The expression level of the antibody molecule can be increased by vector amplification (reviewed,
Bebbington and Hentschel, The use of ve
ctors based on gene amplification fo
r the expression of cloned genes in
mammarian cells in DNA cloning, Vol. 3
. (See Academic Press, New York, 1987). If the marker in the vector system expressing the antibody is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is linked to the antibody gene, antibody production is also increased (Crouse et al., Mol. Cell. Biol. 3: 257).
(1983)).

A host cell can be co-transfected with two expression vectors of the invention, a first vector encoding a heavy chain derived polypeptide and a second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers that allow equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used, which can encode and express both heavy and light chain polypeptides. In such situations, a light chain should be placed in front of the heavy chain to avoid excessive toxic free heavy chains (Pr
audifoot, Nature 322: 52 (1986); Kohler, P
roc. Natl. Acad. Sci. USA 77: 2197 (1980))
. Coding sequences for heavy and light chains can include cDNA or genomic DNA.

Once the antibody molecule of the invention is produced by an animal, chemically synthesized, or recombinantly expressed, any method for purification of immunoglobulin molecules known in the art, For example, chromatography (eg, ion exchange,
It can be purified by affinity (especially by protein A followed by affinity for specific antigens, and size column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification.
Furthermore, the antibodies or fragments thereof of the invention can be fused to heterologous polypeptide sequences as described herein or otherwise known in the art to facilitate purification.

The present invention relates to a polypeptide of the invention (or a portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90,
Or 100 amino acids), recombinantly fused or chemically conjugated (including both covalent and non-covalent bonds) to produce a fusion protein. This fusion need not be direct, but can occur through a linker sequence. This antibody is a polypeptide of the invention (or a portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70,
80, 90, or 100 amino acids). For example, either in vitro or in vivo, the polypeptide of the invention may be directed against a particular cell type by fusing or binding the polypeptide of the invention to an antibody specific for a particular cell surface receptor. To target
Antibodies can be used. Antibodies that are fused or conjugated to the polypeptides of the invention can also be used in in vitro immunoassays and purification methods using methods known in the art. For example, Harbor et al., Supra, and PCT Publication WO 93.
/ 21232; EP 439,095; Naramura et al., Immunol. L
ett. 39: 91-99 (1994); U.S. Pat. No. 5,474,981; G
illies et al., PNAS 89: 1428-1432 (1992); Fell.
Et al. Immunol. 146: 2446-2452 (1991). These are incorporated by reference in their entirety.

The invention further includes a composition comprising a polypeptide of the invention fused or conjugated to a domain other than the variable region of an antibody. For example, a polypeptide of the invention can be fused or conjugated to an Fc region of an antibody, or a portion thereof. The portion of this antibody fused to the polypeptide of the present invention consists of the constant region, hinge region, CH1 domain, C
It can include the H2 domain, and the CH3 domain, or any combination of all or part of the domain. These polypeptides can also be fused or conjugated to the above antibody portions to form multimers. For example, an Fc portion fused to a polypeptide of the present invention can form a dimer through a disulfide bond between the Fc portions. Higher multimeric forms can be made by fusing polypeptides to portions of IgA and IgM. Methods for fusing or linking the polypeptides of the present invention to an antibody moiety are known in the art. For example, U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,04.
No. 6; No. 5,349,053; No. 5,447,851; No. 5,112
, 946; EP 307,434; EP 367,166; PCT publication WO9
6/04388; WO 91/06570; Ashkenazi et al., Proc. N
atl. Acad. Sci. USA 88: 10535-10539 (1991
Zheng et al., J. et al. Immunol. 154: 5590-5600 (199
5); and Vil et al., Proc. Natl. Acad. Sci. USA 89
: 11337-11341 (1992) (the above references are incorporated by reference in their entirety).

As discussed above, a polypeptide corresponding to a polypeptide, polypeptide fragment, or variant of SEQ ID NO: Y is known to increase the in vivo half-life of the polypeptide or in the art. Can be fused or conjugated to the antibody portion described above for use in an immunological assay using the method. further,
A polypeptide corresponding to SEQ ID NO: Y is fused or bound to the above antibody portion,
Purification can be facilitated. One reported example describes a chimeric protein consisting of the first two domains of a human CD4 polypeptide and various domains of the constant region of a mammalian immunoglobulin heavy or light chain (EP 394 , 8
27; Traunecker et al., Nature 331: 84-86 (1988).
)). A polypeptide of the invention, fused or conjugated to an antibody having a disulfide-linked dimeric structure (due to IgG) also binds to other molecules than the monomer secreted protein or protein fragment alone and It may be more efficient to neutralize (Funtoulakis et al., J. Biochem. 270
: 3958-3964 (1995)). In many cases, the Fc portion of the fusion protein is beneficial in therapy and diagnosis, and can thus result, for example, in improved pharmacokinetic properties (EP A 232,262). Alternatively, it may be desirable to delete the Fc portion after the fusion protein has been expressed, detected and purified. For example, when a fusion protein is used as an antigen for immunization, the Fc portion can interfere with therapy and diagnosis. For example, in drug discovery, h
Human proteins such as IL-5 have been fused with the Fc moiety for the purpose of high-throughput screening assays to identify antagonists of hIL-5 (Bennett et al., J. Molecular Recognition).
n 8: 52-58 (1995); Johanson et al., J. Biol. Biol. Che
m. 270: 9459-9471 (1995)).

Furthermore, the antibodies or fragments thereof of the invention can be fused to a marker sequence, such as a peptide that facilitates purification. In a preferred embodiment, the marker amino acid sequence is inter alia a hexa-histidine peptide (eg pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chats).
tags provided in Worth, CA, 91111), and many of these marker amino acid sequences are commercially available. For example, Gentz et al., Proc
. Natl. Acad. Sci. USA 86: 821-824 (1989), hexa-histidine provides convenient purification of the fusion protein. Another peptide tag useful for purification is the “HA” tag corresponding to an epitope from influenza hemagglutinin protein (Wilson et al., Ce
ll37: 767 (1984)), and “flag” tags, but is not so limited.

The invention further includes an antibody or fragment thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically, for example, to monitor tumor development or progression as part of a clinical testing procedure (eg, to determine the efficacy of a given treatment regimen). Detection can be facilitated by linking the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions . This detectable substance is an antibody (or fragment thereof)
In contrast, they can be linked or conjugated either directly or indirectly via a mediator using techniques known in the art, such as linkers known in the art. See, for example, US Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β
Galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin /
Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol Examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive materials include 125I, 131I, 111In or 99Tc.

In addition, the antibody or fragment thereof can be conjugated to a therapeutic moiety (eg, a cytotoxin (eg, cytostatic or cytocidal agent), therapeutic agent or radioactive metal ion (eg, α-emitter—eg, 213Bi)). Can be combined. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D,
Ethidium bromide, emetine, mitomycin, etoposide, teniposide (ten
oposide), vincristine, vinblastine, colchicine (colch)
icin), doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol,
And puromycin, and analogs or homologs thereof. Therapeutic agents include antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine (5-f
urourolacil decarbazine), alkylating agents (eg,
Mechlorethamine, thioepa chlorambucil, melphalan,
Carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (eg Previously daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and antimitotic agents (eg, vincristine and vinblastine) ), But
It is not limited to them.

The conjugates of the invention can be used to modify a given biological response, and the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety can be a protein or polypeptide having the desired biological activity. Such proteins include, for example, toxins (eg, abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin); proteins (eg,
Tumor necrosis factor, a-interferon, β-interferon, nerve growth factor,
Platelet-derived growth factor, tissue plasminogen activator, apoptotic drug (eg, TNF-α, TNF-β, AIM I (International Publication No. WO 97/33899)
No.), AIM II (see WO 97/34911), Fas ligand (Takahashi et al., Int. Immunol. 6).
: 1567-1574 (1994)), VEGI (International Publication No. WO99 / 231)
05)), thrombotic or anti-angiogenic agents (eg angiostatin or endostatin); or biological response modifiers (eg lymphokines, interleukin-1 ("IL-1")) , Interleukin-2 ("IL
-2 "), interleukin-6 (" IL-6 "), granulocyte macrophage colony stimulating factor (" GM-CSF "), granulocyte colony stimulating factor (" G-CSF ")
), Or other growth factors).

The antibody can also be attached to a solid support, which is particularly useful for immunoassay or purification of the target antigen. Such solid supports include, but are not limited to glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for coupling such therapeutic moieties to antibodies are well known, eg, Arnon
Et al., “Monoclonal Antibodies For Immunot.
targeting Of Drugs In Cancer Therapy "
Monoclonal Antibodies And Cancer The
rapy, Reisfeld et al. (ed.), pages 243-56 (Alan R. Lis).
s, Inc. 1985); Hellstrom et al., “Antibodies F
or Drug Delivery "Controlled Drug Del
ivery (2nd edition), Robinson et al. (ed.), pages 623-53 (Marc)
el Dekker, Inc. 1987); Thorpe, “Antibody.
Carriers Of Cytotoxic Agents In Can
cer Therapy: A Review "Monoclonal Anti
bodies '84: Biological And Clinical Ap
applications, Pinchera et al. (eds.), 475-506 (198)
5); "Analysis, Results, And Future Pros
Detective Of The Therapeutic Use Of Ra
Diolabeled Antibody In Cancer Therap
y ”Monoclonal Antibodies For Cancer D
detection And Therapy, Baldwin et al. (edition), 303
-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Prop
erties of Antibodies-Toxin Conjugates "
, Immunol. Rev. 62: 119-58 (1982).

Alternatively, the antibody is conjugated to a secondary antibody and the heterologous conjugate of antibody (as described by Segal in US Pat. No. 4,676,980, which is incorporated by reference herein in its entirety) ( heteroconjugate).

An antibody with or without a therapeutic moiety that binds to the antibody administered alone or in combination with cytotoxic factors and / or cytokines can be used as a therapeutic agent.

(Immunophenotyping)
The antibodies of the invention can be utilized for immunophenotyping of cell lines and biological samples. The translation products of the genes of the present invention may be useful as cell-specific markers or, more particularly, as cell markers that are differentially expressed at various stages of differentiation and / or maturation of a particular cell type. . Monoclonal antibodies directed against a specific epitope, or combination of epitopes, allow screening of cell populations that express the marker. Various techniques can be utilized with monoclonal antibodies to screen populations of cells that express the marker, and include magnetic separation using antibody-coated magnetic beads, solid matrix (ie, “Panning” using antibodies attached to the plate), as well as flow cytometry (eg, US Pat. No. 5,985,6).
60; and Morrison et al., Cell, 96: 737-49 (1999).
)).

These techniques allow for hematological malignancies (ie, minimal residual disease (MRD) in patients with acute leukemia).
And “non-self” in transplantation to prevent graft-versus-host disease (GVHD)
Allows screening of specific populations of cells as can be found with the cells. Alternatively, these techniques allow the screening of hematopoietic stem and progenitor cells that can undergo proliferation and / or differentiation as can be found in human umbilical cord blood.

(Antibody binding assay)
The antibodies of the present invention can be assayed for immunospecific binding by any method known in the art. Immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems. This assay system uses techniques such as the following: Western blot, radioimmunoassay, ELI, to name a few
SA (enzyme-linked immunosorbent assay), “sandwich” immunoassay, immunoprecipitation assay, sedimentation reaction, gel diffusion sedimentation reaction, immunodiffusion assay, aggregation assay, complement binding assay, immunoradiometric assay, fluorescence immunoassay, Protein A
Immunoassay. Such assays are routine and well known in the art (
For example, Ausubel et al. (Ed.), 1994, Current Protocol.
ls in Molecular Biology, Volume 1, John Wil
ey & Sons Inc. , New York. Which is incorporated herein by reference in its entirety). A typical immunoassay is briefly described below (but is not intended as a limitation).

Immunoprecipitation protocols generally use RIPA buffer (1% NP-40 or Tr
iton X-100, 1% sodium deoxycholate, 0.1% SDS, 0
. 15M NaCl, 0.01M sodium phosphate (pH 7.2), 1% Tra
lysing a population of cells in a lysis buffer supplemented with protein phosphatases and / or protease inhibitors (eg, EDTA, PMSF, aprotinin, sodium vanadate), such as Adding to the cell lysate, incubating the protein A and / or protein G sepharose beads for about 1 hour or more at 4 ° C. Including washing the beads with lysis buffer and resuspending the beads in SDS / sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed, for example, by Western blot analysis. Those skilled in the art are familiar with parameters that can be modified to increase the binding of the antibody to the antigen and reduce the background (eg, pre-washing the cell lysate with Sepharose beads). For further discussion on the immunization sedimentation protocol, see Ausubel et al. (Ed.), 1994, Current Protocols.
in Molecular Biology, Vol. 1. John Wile
y & Sons, Inc. , New York (10.16.1).

Western blot analysis generally involves preparing a protein sample, electrophoresis of the protein sample on a polyacrylamide gel (eg, 8% to 20% SDS-PAGE depending on the molecular weight of the antigen), and the protein sample from the polyacrylamide gel to the membrane. (E.g. transfer to nitrocellulose, PVDF or nylon), block membrane in blocking solution (e.g. PBS containing 3% BSA or non-fat milk), wash membrane (e.g. PBS-Tween20) Washing step, blocking membrane using primary antibody (target antibody) diluted in blocking buffer, washing membrane in washing buffer, enzyme diluted in blocking buffer Substrate (for example, Horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., secondary antibody conjugated to 32P or @ 125 I) (
This includes the steps of blocking the membrane with a primary antibody (eg, an anti-human antibody), washing the membrane in a wash buffer, and detecting the presence of the antigen. Those skilled in the art are familiar with parameters that can be modified to increase the detected signal and reduce background noise. For further discussion on Western blot protocols, see, eg, Aus
ubel et al. (ed.), 1994, Current Protocols in M
molecular biology, Vol. 1. John Wiley & So
ns, Inc. , New York (10.8.1).

ELISAs are intended for preparing antigens, coating the wells of a 96-well microtiter plate with the antigens, and of interest bound to a detectable compound such as an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase). Adding an antibody to the well and incubating for a period of time and detecting the presence of the antigen. In an ELISA, the antibody of interest need not be bound to a detectable compound; instead, a second antibody (recognizing the antibody of interest) bound to the detectable compound can be added to the well. Further, instead of coating the well with the antigen, the antibody can be coated to the well. In this case, a second antibody conjugated to a detectable compound can be added following the addition of the antigen of interest to the coated well. One skilled in the art can appreciate with respect to parameters that can be modified to increase the detected signal, and other variations of ELISA known in the art. For further discussion on ELISA, see, for example, Ausubel et al., 1994, Cur.
rent Protocols in Molecular Biology,
Volume 1, John Wiley & Sons, Inc. , New York,
See 11.2.1.

The binding affinity of the antibody to the antigen and the off-rate of the antibody-antigen interaction (of
f-rate) can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay, which comprises incubation of a labeled antigen (eg, 3H or 125I) with an antibody of interest in the presence of increasing amounts of unlabeled antigen, and labeling. Detection of antibodies bound to the antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rate can be determined from data by Scatchard plot analysis. Competition with the second antibody can also be determined using a radioimmunoassay. In this case, the antigen is labeled in the presence of increasing amounts of unlabeled second antibody (eg 3H or 125I).
Incubated with the antibody of interest bound to

(Therapeutic use)
The present invention further relates to antibody-based therapies, which comprise treating an antibody of the present invention with an animal, preferably a mammal, to treat one or more disclosed diseases, disorders, or conditions, and Most preferably, it comprises the step of administering to a human patient. Therapeutic compounds of the invention include the antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding the antibodies of the invention (described herein). Including, but not limited to, fragments, analogs and derivatives thereof and anti-idiotype antibodies. An antibody of the invention includes a disease, disorder or condition associated with aberrant expression and / or activity of a polypeptide of the invention (although any one or more of the diseases, disorders or conditions described herein). (But not limited to) can be used to treat, inhibit or prevent. Treatment and / or prevention of diseases, disorders or conditions associated with aberrant expression and / or activity of the polypeptides of the present invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Not.
The antibodies of the present invention are known in the art or can be provided in pharmaceutically acceptable compositions as described herein.

One summary of how the antibodies of the invention can be used therapeutically is local or systemic in the body, or mediated by, for example, complement (CDC) or effector cells (ADCC). Due to the direct cytotoxicity of the antibody
It includes conjugating a polynucleotide or polypeptide of the present invention. Some of these approaches are described in more detail below. Given the teaching provided herein, one of ordinary skill in the art will be able to determine how to use the antibodies of the present invention for diagnostic, monitoring, or therapeutic purposes without undue experimentation. Recognize.

The antibodies of the present invention may be used, for example, for other monoclonal or chimeric antibodies, or lymphokines or hematopoietic growth factors (eg, IL-2, IL-3) that serve to increase the number or activity of effector cells that interact with the antibody. And IL
-7 etc.) and can be advantageously used.

The antibodies of the invention can be administered alone or in combination with other types of treatments (eg, radiation therapy, chemotherapy, hormone therapy, immunotherapy and anti-tumor agents).
In general, administration of a species-origin or species-reactive product that is the same species as the patient's species (in the case of antibodies) is preferred. Thus, in a preferred embodiment, a human antibody,
Fragment derivatives, analogs, or nucleic acids are administered to human patients for treatment or prevention.

High affinity and / or potent against the polypeptides or polynucleotides of the present invention, both for immunoassays relating to the polynucleotides or polypeptides of the present invention (including fragments thereof) and for the treatment of disorders associated therewith. Preferably, in vivo inhibitory and / or neutralizing antibodies, fragments thereof, or regions thereof are used. Such antibodies, fragments or regions preferably have affinity for the polynucleotides or polypeptides of the invention (including fragments thereof). Preferred binding affinities are 5 × 10 ⁻² M, 10 ⁻² M, 5 × 10 ⁻³ M, 10 ⁻³ M, 5 × 10 ⁻⁴ M,
10 ⁻⁴ M, 5 × 10 ⁻⁵ M, 10 ⁻⁵ M, 5 × 10 ⁻⁶ M, 10 ⁻⁶ M, 5 × 10 ⁻⁷ M,
10 ⁻⁷ M, 5 × 10 ⁻⁸ M, 10 ⁻⁸ M, 5 × 10 ⁻⁹ M, 10 ⁻⁹ M, 5 × 10 ⁻¹⁰ M
10 ⁻¹⁰ M, 5 × 10 ⁻¹¹ M, 10 ⁻¹¹ M, 5 × 10 ⁻¹² M, 10 ⁻¹² M, 5 × 1
0 ⁻¹³ M, 10 ⁻¹³ M, 5 × 10 ⁻¹⁴ M, 10 ⁻¹⁴ M, 5 × 10 ⁻¹⁵ M, and 10 ⁻
Mention may be made of binding affinities with dissociation constants smaller than ¹⁵ M, ie Kd.

(Gene therapy)
In certain embodiments, a nucleic acid comprising a sequence encoding an antibody or functional derivative thereof is used to treat, inhibit or prevent a disease or disorder associated with aberrant expression and / or activity of a polypeptide of the invention. Administered for gene therapy purposes. Gene therapy refers to treatment performed by administering to a subject an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein, which mediates a therapeutic effect.

Any method for gene therapy available in the art can be used in accordance with the present invention. An exemplary method is described below.

For a general review of methods of gene therapy, see Goldspiel et al., Cli.
nical Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstos
hev, Ann. Rev. Pharmacol. Toxicol. 32: 573
-596 (1993); Mulligan, Science 260: 926.
932 (1993); and Morgan and Anderson, Ann.
Rev. Biochem. 62: 191-217 (1993); May, TIB
See TECH 11 (5): 155-215 (1993). Methods commonly known in the recombinant DNA art that can be used are Ausubel.
(Eds.), Current Protocols in Molecular
Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Express.
ion, A Laboratory Manual, Stockton Pre
ss, NY (1990).

In a preferred aspect, the compound contains a nucleic acid sequence encoding an antibody, said nucleic acid sequence being part of an expression vector that expresses the antibody, or fragment or chimeric protein thereof, or heavy or light chain thereof in a suitable host. It is. In particular, such nucleic acid sequences have a promoter operably linked to the antibody coding region, said promoter being inducible or constitutive, and optionally tissue specific. In another specific embodiment, a nucleic acid molecule is used that is flanked by a region encoding the antibody and any other desired sequence that promotes homologous recombination at the desired site in the genome. Provides chromosomal expression of the nucleic acid encoding (Koller and Smithies, Proc
. Natl. Acad. Sci. USA 86: 8932-8935 (1989
Zijlstra et al., Nature 342: 435-438 (1989).
). In certain embodiments, the expressed antibody molecule is a single chain antibody; or the nucleic acid sequence comprises a sequence encoding both the heavy and light chains of the antibody or a fragment thereof.

Delivery of the nucleic acid to the patient can be direct (in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector) or indirectly (in which case the cells are first transformed with the nucleic acid in vitro, And then transplanted into the patient). These two approaches are respectively known as in vivo gene therapy or as ex vivo gene therapy.

In certain embodiments, the nucleic acid sequence is administered directly in vivo, where the nucleic acid sequence is expressed to produce the encoded product. This can be done by any of a number of methods known in the art, such as by constructing them as part of a suitable nucleic acid expression vector and administering it to be intracellular (eg, defective By infection with sex or attenuated retroviruses or other viral vectors (see US Pat. No. 4,980,286), or
Liposomes by direct injection of naked DNA or by the use of microparticle bombardments (eg gene guns; Biolistic, Dupont) or by coating with lipids or cell surface receptors or by transfecting drugs; Bind it to a ligand that undergoes receptor-mediated endocytosis, either by encapsulation in microparticles or microcapsules, or by administering them in combination with peptides known to enter the nucleus. By administration (see, eg, Wu and Wu, J. et al.
Biol. Chem. 262: 4429-4432 (1987)).
(Which can be used to target cell types that specifically express the receptor). In another embodiment, a nucleic acid-ligand complex may be formed, wherein the ligand is a fusogenic that disrupts endosomes.
) Contain a viral peptide so that the nucleic acid avoids lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell-specific uptake and expression by targeting specific receptors (eg, PCT Publication No. WO 92/06180; No. 22635; No. WO 92/20316; No. WO 93/14188, No. WO 93/202
(See 21). Alternatively, the nucleic acid can be introduced into the cell and incorporated into host cell DNA for expression by homologous recombination (Koller and Smithes, Proc. Natl. Acad. Sci. USA 86
: 8932-8935 (1989); Zijlstra et al., Nature 34.
2: 435-438 (1989)).

In certain embodiments, viral vectors that contain nucleic acid sequences encoding an antibody of the invention are used. For example, retroviral vectors can be used (Mi
ller et al., Meth. Enzymol. 217: 581-599 (1993)
checking). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and the correct integration into the host cell DNA. Nucleic acid sequences encoding antibodies used in gene therapy are cloned into one or more vectors, which facilitates gene delivery into the patient. For further details on retroviral vectors, see Boesen et al., Bio
therapy 6: 291-302 (1994), which describes the use of retroviral vectors to deliver the mdrI gene to hematopoietic stem cells to make them more resistant to chemotherapy. Can be issued. Other references describing the use of retroviral vectors in gene therapy are:
: Clowes et al. Clin. Invest. 93: 644-651 (19
94); Kiem et al., Blood 83: 1467-1473 (1994); S
almons and Gunzberg, Human Gene Therapy
4: 129-141 (1993); and Grossman and Wils.
on, Curr. Opin. in Genetics and Devel. 3
: 110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are particularly attractive vehicles for delivering genes to the respiratory epithelium. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage that they can infect non-dividing cells. Kozarsky and Wilson, Current Opin
ion in Genetics and Development 3:49
9-503 (1993) provides an overview of adenovirus-based gene therapy.
Bout et al., Human Gene Therapy 5: 3-10 (1994)
) Demonstrated the use of adenoviral vectors to transfer genes to the rhesus monkey airway epithelium. Another example of the use of adenovirus in gene therapy is Ro
senfeld et al., Science 252: 431-434 (1991); R
osenfeld et al., Cell 68: 143-155 (1992);
rangeli et al., J. MoI. Clin. Invest. 91: 225-234 (19
93); PCT Publication No. WO 94/12649; and Wang et al., Gene.
Therapy 2: 775-783 (1995). In a preferred embodiment, adenoviral vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 2).
04: 289-300 (1993); US Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer involves the transfer of a selectable marker to the cells. The cells are then placed in selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to the patient.

In this embodiment, the nucleic acid is introduced into the cell prior to in vivo administration of the resulting recombinant cell. Such introduction can be performed by any method known in the art, including but not limited to: viral vectors containing transfection, electroporation, microinjection, nucleic acid sequences. Or infection with bacteriophage vectors, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for introducing foreign genes into cells (eg, Loeffler and Behr, Meth. Enzymol. 217: 599-618 (1993).
Cohen et al., Meth. Enzymol. 217: 618-644 (19
93); Cline, Pharmac. Ther. 29: 69-92m (198
See 5)), and can be used according to the present invention if the necessary developmental and physiological functions of the recipient cell are not destroyed. This technique should provide for a stable transfer of the nucleic acid into the cell so that the nucleic acid can be expressed by the cell and is preferably heritable and expressible by the cell's progeny. is there.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (eg, hematopoietic stem cells or hematopoietic progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which the nucleic acid can be introduced for gene therapy purposes include any desired available cell type and include, but are not limited to: epithelial cells, endothelial cells, keratinocytes, fibroblasts, Muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem cells or progenitor cells, in particular hematopoietic stem cells or hematopoietic cells Progenitor cells (eg, cells obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.).

In preferred embodiments, the cells used for gene therapy are autologous to the patient.

In embodiments where a recombinant cell is used in gene therapy, the nucleic acid sequence encoding the antibody is introduced into the cell such that the nucleic acid sequence can be expressed by the cell or their progeny, and then the recombinant cell is therapeutically Administered in vivo for effect. In certain embodiments, stem cells or progenitor cells are used. Any stem and / or progenitor cell that can be isolated in vitro and stored in vitro can potentially be used according to this embodiment of the invention (eg, P
CT Publication No. WO 94/08598: Temple and Anderson,
Cell 71: 973-985 (1992); Rheinwald, Meth.
. Cell Bio. 21A: 229 (1980); and Pittelko
w and Scott, Mayo Clinic Proc. 61: 771 (19
86)).

In certain embodiments, the nucleic acid introduced for gene therapy purposes contains an inducible promoter operably linked to the coding region so that the expression of the nucleic acid is present in the presence or absence of a suitable transcription inducer. It can be controlled by controlling its presence.

(Demonstration of therapeutic or prophylactic activity)
The compounds or pharmaceutical compositions of the invention are preferably tested for the desired therapeutic or prophylactic activity in vitro prior to use in humans and then in vivo. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of the compound on cell lines or patient tissue samples. The effect of the compound or composition on the cell line and / or tissue sample can be determined utilizing techniques known to those of skill in the art, including but not limited to rosette formation assays and cell lysis assays. In vitro assays that can be used to determine whether administration of a particular compound is indicated according to the present invention include in vitro cell culture assays, in which a patient tissue sample is grown in culture and the compound Or otherwise a compound is administered and the effect of such compound on the tissue sample is observed.

(Therapeutic / prophylactic administration and composition)
The present invention provides methods of treatment, inhibition and prevention by administration of an effective amount of a compound or pharmaceutical composition of the present invention, preferably an antibody of the present invention, to a subject. In preferred aspects, the compound is substantially purified (eg, substantially free of substances that limit its effect or cause undesirable side effects). The subject is preferably
Animals include but are not limited to animals such as cows, pigs, horses, chickens, cats, dogs and the like, and are preferably mammals, and most preferably humans.

Formulations and methods of administration that can be used when the compound comprises a nucleic acid or immunoglobulin are described above; further suitable formulations and routes of administration are selected from those described herein below. obtain.

Various delivery systems are known and can be used to administer the compounds of the invention (eg, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated endos) Cytosis (eg, Wu and Wu, J. Biol. Chem. 262: 4429-4432 (
(1987)), construction of nucleic acids as part of retroviruses or other vectors, etc.). Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition can be administered by any convenient route (eg, by infusion or bolus injection, by absorption through epithelial or mucosal linings (eg, oral mucosa, rectal mucosa, intestinal mucosa, etc.), and others. Can be administered together with other biologically active agents. Administration can be systemic or local. In addition, the pharmaceutical compounds or compositions of the present invention may be administered by any suitable route (including intraventricular and intrathecal injection; intraventricular injection, for example, an intraventricular catheter attached to a reservoir, such as an Ommaya reservoir. It may be desirable to introduce it into the central nervous system. Pulmonary administration can also be used, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In certain embodiments, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this is not meant to be limiting, but for example, during surgery By injection, topical application (eg in combination with a post-surgical wound dressing), by injection, by catheter, by suppository, or an implant (this implant comprises a membrane or fiber, such as a sialastic membrane) Porous, non-porous, or gelatin-like material). Preferably, when administering a protein of the invention comprising an antibody,
Care must be taken to use materials that do not absorb protein.

In another embodiment, the compound or composition can be delivered into vesicles, particularly liposomes (Langer, Science 249: 1527-1533 (
1990); Treat et al., Liposomes in the Therap.
y of Infectious Disease and Cancer, L
opez-Berstein and Fiddler (edition), Liss, New
York, pages 353-365 (1989); Lopez-Berstein,
(See pages 317-327 of the same book; broadly see the same book).

In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump can be used (Lang
er, (supra); Sefton, CRC Crit. Ref. Biomed. E
ng. 14: 201 (1987); Buchwald et al., Surgery 88.
: 507 (1980); Saudek et al., N .; Engl. J. et al. Med. 321:
574 (1989)). In another embodiment, polymeric materials can be used (Medical Applications of Control).
led Release, Langer and Wise (ed.), CRC Pre
s. , Boca Raton, Florida (1974); Control
ed Drug Bioavailability, Drug Product
Design and Performance, Smolen and Bal
l (eds.), Wiley, New York (1984); Ranger and P
eppas, J. et al. Macromol. Sci. Rev. Macromol. Ch
em. 23:61 (1983); Levy et al., Science 2
28: 190 (1985); During et al., Ann. Neurol. 25: 3
51 (1989); Howard et al., J. MoI. Neurosurg. 71: 105 (
(1989)). In yet another embodiment, the controlled release system can be placed near the therapeutic target, i.e., the brain, and thus requires only a portion of the systemic dose (e.g., Goodson, Medical Application).
ions of Controlled Release (supra), Volume 2,
115-138 (1984)).

Other controlled release systems are discussed in a review by Langer (Science 249: 1527-1533 (1990)).

In a specific embodiment, where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid is constructed as part of a suitable nucleic acid expression vector and administered so that it is intracellular. (Eg, by use of retroviral vectors (see US Pat. No. 4,980,286)) or by direct injection or microparticle bombardment (eg, gene gun; Biolis
tic, Dupont) or by coating with lipids or cell surface receptors or transfection agents, or in combination with homeobox-like peptides known to enter the nucleus (eg,
Joliot et al., Proc. Natl. Acad. Sci. USA 88:18
64-1868 (1991)) and the like can be administered in vivo to promote expression of the encoded protein. Alternatively, the nucleic acid can be introduced into the cell and incorporated into the host cell DNA by homologous recombination for expression.

The present invention also provides a pharmaceutical composition. Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” is recognized by federal regulatory authorities or the US government for use in animals, and more particularly in humans, or the US Pharmacopoeia or other Means listed in the generally accepted pharmacopoeia.
The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers include sterile liquids, such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin (eg, peanut oil, soybean oil, mineral oil, sesame oil). It can be. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene , Glycol, water, ethanol and the like. The composition can also include minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are E. W. “Remin” by Martin
gton's Pharmaceutical Sciences ". Such compositions comprise a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the dosage form.

In a preferred embodiment, the composition is formulated as a pharmaceutical composition employed for intravenous administration to humans according to conventional procedures. Typically, compositions for intravenous administration are sterile isotonic aqueous buffer solutions. Where necessary, the composition may also include a solubilizer and a local anesthetic such as lignocaine that relieves pain at the site of the injection. In general, the components are either separated or mixed together in a single dosage form, for example, an ampoule or sachet (s
supplied as a lyophilized powder or a water-free concentrate in a sealed container such as achette). Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, so that the ingredients can be mixed prior to administration,
Ampules of sterile water for injection or saline can be provided.

The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and sodium, potassium, ammonium, calcium, ferric hydroxide, Isopropylamine, triethylamine,
And salts formed with cations such as those derived from 2-ethylaminoethanol, histidine, procaine, and the like.

The amount of a compound of the invention that is effective in this treatment (suppression and prevention of a disease or disorder associated with aberrant expression and / or activity of a polypeptide of the invention) can be determined by standard clinical techniques. In addition, in vitro assays can be used, if necessary, to help identify optimal dosage ranges. The exact dose to be used in the formulation will also depend on the route of administration, and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and the circumstances of each patient. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically from 0.1 mg / kg to 100 mg / kg of the patient's body weight. Preferably, the dosage administered to the patient is between 0.1 mg / kg and 20 mg / kg of the patient's body weight, more preferably 1 mg / kg to 10 mg / kg of the patient's body weight. In general, human antibodies have longer half-lives in the human body than antibodies from other species due to immune responses to foreign polypeptides. Thus, low dosages and infrequent administration of human antibodies is often possible. Furthermore, the dosage and frequency of administration of the antibodies of the invention may be altered (eg,
It can be reduced by enhancing antibody uptake and tissue penetration (eg, into the brain) by lipidation (such as lipidation).

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical composition of the present invention. Notifications in the form prescribed by government agencies regulating the manufacture, use or sale of pharmaceutical or biological products may be optionally accompanied by such containers. This notice
Reflects this agency's approval for manufacture, use or sale for human administration.

(Diagnosis and imaging)
Labeled antibodies that specifically bind to the polypeptide of interest, and derivatives and analogs thereof, are used for diagnostic purposes to treat diseases, disorders associated with abnormal expression and / or activity of the polypeptides of the invention. And / or the condition may be detected, diagnosed or monitored. The present invention provides for the detection of aberrant expression of a polypeptide of interest, which comprises: (a) using one or more antibodies specific for the polypeptide of interest to produce the object in a cell or body fluid of an individual. Assaying expression of the polypeptide of
And (b) comparing the gene expression level with a standard gene expression level, thereby increasing or decreasing the assayed polypeptide gene expression level compared to the standard expression level. Shows abnormal expression.

The present invention provides a diagnostic assay for diagnosing a disorder, the assay comprising:
a) assaying the expression of the polypeptide of interest in a cell or body fluid of an individual using one or more antibodies specific for the polypeptide of interest; and (b) this gene expression level and standard Comparing the level of gene expression, whereby an increase or decrease in the assayed polypeptide gene expression level compared to its standard expression level indicates a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in an individual's biopsy tissue may indicate a predisposition for the development of the disease or provide a means for detecting the disease before the appearance of actual clinical symptoms Can do. This type of more definitive diagnosis may allow health professionals to use preventive measures, or allow for earlier and aggressive treatment, thereby preventing the development or further progression of cancer.

The antibodies of the invention can be used to assay protein levels in biological samples using classical immunohistological methods known to those skilled in the art (eg, J
alkanen et al. Cell. Biol. 101: 976-985 (198
5); Jalkanen et al. Cell. Biol. 105: 3087-30
96 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).
Suitable antibody assay labels are known in the art and are enzyme labels (eg glucose oxidase); radioisotopes (eg iodine (125I, 125I,
121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc)); luminescent labels (eg, luminol); and fluorescent labels (eg, fluorescein and rhodamine),
As well as biotin.

One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal, and most preferably a human. In one embodiment, diagnosis comprises a) administering to a subject (eg, parenterally, subcutaneously, or intraperitoneally) an effective amount of a labeled molecule that specifically binds to the polypeptide of interest. B) to allow preferential enrichment of the labeled molecule at the site in the subject where the polypeptide is expressed (and to remove unbound labeled molecule to background levels); A) waiting for a time interval after administration; c) determining a background level; and d) detecting a labeled molecule in the subject so that the label exceeds the background level. Detection of the activated molecule indicates that the subject has a particular disease or disorder associated with abnormal expression of the polypeptide of interest. The background level can be determined by a variety of methods including comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

It will be appreciated in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety needed to produce a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected is typically about 5 to 5
It is in the range of 99 mTc of 20 millicuries. The labeled antibody or antibody fragment is then preferentially accumulated at the location of the cell containing the particular protein.
In vivo tumor imaging is described in S.H. W. Burchiel et al., “Immunofar.
macokinetics of Radiolabeled Antibodies
ies and Ther Fragments "(Tumor Image)
ng: The Radiochemical Detection of Ca
ncer Chapter 13, S.C. W. Burchiel and B.M. A. Edited by Rhodes, Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and the mode of administration, the labeled molecules preferentially concentrate at the site of the subject, and unbound labeled molecules are background The time interval after administration that allows it to be cleared to a level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours.
In another embodiment, the time interval after administration is 5-20 days or 5-10 days.

In one embodiment, monitoring a disease or disorder involves repeating a method for diagnosing the disease or disorder (eg, 1 month after initial diagnosis, 6 months after initial diagnosis, 1 year after initial diagnosis). Etc.).

The presence of the labeled molecule can be detected from the patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. One skilled in the art can determine an appropriate method for detecting a particular label. Methods and devices that can be used in the diagnostic methods of the present invention include, but are not limited to, whole body scanning such as computed tomography (CT), proton emission tomography (PET), magnetic resonance imaging (MRI). ), And ultrasonography.

In certain embodiments, the molecule is labeled with a radioisotope and detected from the patient using a radiation responsive surgical instrument (Thurston et al., US Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and detected from the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and detected from a patient using proton emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and magnetic resonance imaging (MRI)
Is detected from the patient.

(kit)
The present invention provides kits that can be used in the above methods. In one embodiment, the kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In certain embodiments, the kits of the invention comprise a substantially isolated polypeptide comprising an epitope. This epitope specifically immunoreacts with the antibody contained in the kit. Preferably, the kit of the present invention further comprises a control antibody that does not react with the target polypeptide. In another specific embodiment, the kit of the invention comprises means for detecting binding of an antibody to a polypeptide of interest (eg, the antibody is a detectable substrate (eg, a fluorescent compound, an enzyme substrate). , Radioactive compounds or luminescent compounds) or secondary antibodies that recognize the primary antibody can be conjugated with a detectable substrate).

In another specific embodiment of the invention, the kit is a diagnostic kit for use in screening sera containing antibodies specific for proliferative and / or cancerous polynucleotides and polypeptides. Such a kit may comprise a control antibody that does not react with the polypeptide of interest. Such a kit can comprise a substantially isolated polypeptide antigen comprising an epitope. This epitope specifically immunoreacts with at least one anti-polypeptide antigen antibody. In addition, such kits comprise means for detecting the binding of the antibody to the antigen (eg, the antibody is a fluorescent compound (eg, fluorescein or rhodamine that can be detected by flow cytometry) and a conjugate. Can be In certain embodiments, the kit can comprise a recombinantly produced polypeptide antigen or a chemically synthesized polypeptide antigen. The polypeptide antigens of the kit can also be attached to a solid support.

In a more specific embodiment, the detection means of the kit comprises a solid support to which the polypeptide antigen is attached. Such a kit may also comprise a non-attached reporter labeled anti-human antibody. In this embodiment, antibody binding to the polypeptide antigen can be detected by binding of the reporter-labeled antibody described above.

In a further embodiment, the present invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the present invention. The diagnostic kit comprises a substantially isolated antibody that specifically immunoreacts with a polypeptide antigen or polynucleotide antigen, and a means for detecting binding of the polynucleotide antigen or polypeptide antigen to the antibody. Prepare. In one embodiment, the antibody is attached to a solid support. In certain embodiments, the antibody can be a monoclonal antibody. This detection means of the kit may comprise a secondary labeled monoclonal antibody. Alternatively or additionally, the detection means may include a labeled competitor antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the method of the present invention. After binding of the specific antigen antibody to this reagent and removal of unbound serum components by washing, the reagent is reacted with a reporter-labeled anti-human antibody to bind the bound anti-antigen onto the solid support. A reporter is bound to this reagent in proportion to the amount of antigen antibody. The reagent is washed again to remove unbound labeled antibody and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme, which is detected by incubating the solid phase in the presence of a suitable fluorescent, luminescent or colorimetric substrate (Sigma, St. Louis, MO). Is done.

In the above assay, the solid surface reagent is prepared by known techniques for attaching protein substances to solid support substances (eg, polymer beads, dipsticks, 96 well plates or filtration materials). These attachment methods generally involve non-specific adsorption of the protein to the support or covalent attachment of the protein (typically free amine groups relative to chemically reactive groups on the solid support (
For example, an activated carboxyl group, a hydroxyl group, or an aldehyde group). Alternatively, streptavidin-coated plates can be used in combination with biotinylated antigen.

Thus, the present invention provides an assay system or kit for performing this diagnostic method. The kit generally comprises a support having a recombinant antigen bound to the surface,
And a reporter-labeled anti-human antibody for detecting anti-antigen antibodies bound to the surface.

(Fusion protein)
Any polypeptide of the invention can be used to produce a fusion protein. For example, a polypeptide of the invention can be used as an antigenic tag when fused to a second protein. An antibody raised against a polypeptide of the invention can be used to indirectly detect a second protein by binding to the polypeptide. Furthermore, since secreted proteins target cell location based on transport signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.

Examples of domains that can be fused to the polypeptides of the invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion need not be direct, but can occur through a linker sequence.

In addition, fusion proteins can also be engineered to improve the characteristics of the polypeptides of the invention. For example, additional amino acids, particularly regions of charged amino acids, can be added to the N-terminus of a polypeptide to improve stability and persistence during purification from host cells or subsequent handling and storage. Peptide moieties can also be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of the polypeptide is a conventional technique well known in the art.

In addition, the polypeptides of the invention (including fragments, and in particular epitopes) are constant domains of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof (
Combined with a portion of the entire domain and both portions thereof)), can give rise to a chimeric polypeptide. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example is human CD4
Describes a chimeric protein consisting of the first two domains of a polypeptide and various domains of the constant region of a mammalian immunoglobulin heavy or light chain (EP A 394,827; Traunecker et al., Nature 3
31: 84-86 (1988)). Fusion proteins with disulfide-linked dimeric structures (due to IgG) can also be more efficient to bind and neutralize other molecules than monomer secreted proteins or protein fragments alone (Fontoulakis et al. J. Biochem. 270: 3.
958-3964 (1995)). Polynucleotides comprising or consisting of nucleic acids encoding these fusion proteins are also encompassed by the present invention.

Similarly, EP-A-O 464 533 (Canadian counterpart patent 2045869).
No.) discloses a fusion protein comprising various portions of the constant region of an immunoglobulin molecule together with another human protein or portion thereof. In many cases, the Fc portion of the fusion protein is beneficial in therapy and diagnosis, and can thus result, for example, in improved pharmacokinetic properties (EP-A 0232 262). Alternatively, it may be desirable to delete the Fc portion after the fusion protein has been expressed, detected and purified. For example, when a fusion protein is used as an antigen for immunization, the Fc portion can interfere with therapy and diagnosis. For example, in drug discovery, human proteins such as hIL-5 have been fused with Fc moieties for the purpose of high throughput screening assays to identify antagonists of hIL-5 (D. Bennett et al., J. Molecular Reco
gnition 8: 52-58 (1995); Johanson et al.
Biol. Chem. 270: 9459-9471 (1995)).
.

Furthermore, the polypeptides of the invention can be fused to a marker sequence (eg, a peptide that facilitates purification of the fused polypeptide). In a preferred embodiment, the marker amino acid sequence is inter alia a hexa-histidine peptide (eg p
QE vector (QIAGEN, Inc., 9259 Eton Avenue,
Tags provided in Chatsworth, CA, 91111).
Many of these marker amino acid sequences are commercially available. For example, Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1
989), hexa-histidine provides convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from influenza hemagglutinin protein (Wi
lson et al., Cell 37: 767 (1984)).

Accordingly, any of these above fusions can be engineered using the polynucleotides or polypeptides of the invention.

(Vector, host cell, and protein production)
The invention also relates to vectors, host cells, and recombinant polypeptide production comprising the polynucleotides of the invention. For example, the vector can be a phage vector, a plasmid vector, a viral vector, or a retroviral vector. Retroviral vectors can be replication competent or replication defective. In the latter case, viral propagation is generally complementary (complement).
occurs only in host cells.

The polynucleotide can be linked to a vector containing a selectable marker for propagation in the host. In general, plasmid vectors are introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, the viral vector can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

Polynucleotide inserts are suitable promoters (eg, phage λPL promoter, E. coli lac promoter, trp promoter, phoA promoter and tac promoter, SV40 early promoter and SV40 late promoter, and promoter of retroviral LTR, to name a few. ) Should be operatively coupled to. Other suitable promoters are known to those skilled in the art. The expression construct further includes sites for transcription initiation, transcription termination,
And in the transcription region, contains a ribosome binding site for translation. The coding portion of the transcript expressed by the construct is preferably initially a translation initiation codon and a termination codon (UA) appropriately located at the end of the polypeptide to be translated.
A, UGA or UAG).

As indicated, the expression vector preferably includes at least one selectable marker. Such markers include dihydrofolate reductase, G418, or neomycin resistance genes for eukaryotic cell culture, and E. coli. Contains tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of suitable hosts are bacterial cells (eg, E.c.
oli, Streptomyces and Salmonella typhim
urium cells); fungal cells such as yeast cells (eg, Saccharomy
ces cerevisiae or Pichia pastoris (ATC
C accession number 201178)); Drosophilia S2 and Spodo
including but not limited to insect cells such as ptera Sf9 cells; animal cells such as CHO cells, COS cells, 293 cells, and Bowes melanoma cells; and plant cells. Appropriate culture media and conditions for the above-described host cells are known in the art.

Among the preferred vectors for use in bacteria are pQE70, pQE6
0 and pQE-9 (available from QIAGEN, Inc.); pBluesc
rrip vector, Pagescript vector, pNH8A, pNH16
a, pNH18A, pNH46A (Stratagene Cloning S)
systems, Inc. And ptrc99a, pKK223
-3, pKK233-3, pDR540, pRIT5 (Pharmacia B
iotech, Inc. Available). Among the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (S
available from tratagene); and pSVK3, pBPV, pMSG
And pSVL (available from Pharmacia). Preferred expression vectors for use in yeast systems are pYES2, pYD1, pTEF1 / Z.
eo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalph, pP
IC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K
, PPIC9K, and PAO815 (all Invitrogen, Carl
but available from bad, CA). Other suitable vectors will be readily apparent to those skilled in the art.

Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DE
It can be effected by AE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other methods. Such a method is described by Davis et al., Basic Method.
It is described in many standard laboratory manuals such as ds In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the invention may in fact be expressed by host cells that lack the recombinant vector.

The polypeptides of the present invention can be prepared by ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. It can be recovered from the recombinant cell culture and purified by well-known methods including. Most preferably, high performance liquid chromatography (“HPLC”)
Is used for purification.

The polypeptides of the invention, and preferably secreted forms, can also be recovered from: purified products from natural sources, including body fluids, tissues and cells, whether isolated directly or cultured The product of a chemical synthesis procedure; and
Products produced recombinantly from prokaryotic or eukaryotic hosts, including, for example, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells. Depending on the host used for the recombinant production procedure, the polypeptides of the present invention may or may not be glycosylated. Furthermore, the polypeptides of the present invention may also contain an initial modified methionine residue in some cases as a result of host-mediated processes. Thus, it is generally well known in the art that the N-terminal methionine encoded by the translation initiation codon is efficiently removed from any post-translational protein in all eukaryotic cells. N-terminal methionine is also effectively removed in most prokaryotes in most proteins, but for some proteins this prokaryotic removal process depends on the nature of the amino acid to which the N-terminal methionine is covalently attached. And inefficient.

In one embodiment, the yeast Pichia pastoris is used to express a polypeptide of the invention in a eukaryotic system. Pichia
pastoris is a methylotrophic yeast that can metabolize methanol as its sole carbon source. The main step in the methanol metabolic pathway is to oxidize methanol to formaldehyde using O ₂ . This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia p
astoris is somewhat due to the relatively low affinity of alcohol oxidase for O _2, must generate high levels of alcohol oxidase. Thus, in a growth medium with methanol as the main carbon source, 2
The promoter region of one alcohol oxidase gene (AOX1) is very active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene constitutes up to approximately 30% of the total soluble protein in Pichia pastoris. Ellis, S.M. B. Et Mo
l. Cell. Biol. 5: 1111-21 (1985); Koutz, P .;
J. et al. Yeast 5: 167-77 (1989); Tschop, J. et al. F
. Et al., Nucl. Acids Res. 15: 3859-76 (1987). Thus, under transcriptional control of all or part of the AOX1 regulatory base sequence, heterologous coding sequences (eg, polynucleotides of the invention) are expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol. .

In one example, the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention in a Picea yeast system essentially as described below, as described herein. Used: “Pichia Protocols: Methods in Molecular Biology”, D.C. R. Higgins and J.H. Edited by Cregg, The Humana Press, Totowa, NJ, 1998. This expression vector, Pichia pastoris alkaline phosphatase located upstream of the multiple cloning site (PHO) secretory signal peptide (i.e., leader peptide) by virtue of a strong AOX1 promoter linked to the expression and secretion of protein of the present invention enable.

Many other yeast vectors will readily signal that the proposed expression construct is properly positioned for transcription, translation, secretion (if desired), etc., as will be readily appreciated by those skilled in the art. PPIC9 as long as it provides in-frame AUG)
K (for example, pYES2, pYD1, pTEF1 / Zeo, pYES2 / GS,
pPICZ, pGAPZ, pGAPZα, pPIC9, pPIC3.5, pHI
L-D2, pHIL-S1, pPIC3.5K, and PAO815) can be used.

In another embodiment, high level expression of a heterologous coding sequence (such as a polynucleotide of the present invention) clones a heterologous polynucleotide of the present invention into an expression vector (such as pGAPZ or pGAPZα). And the yeast culture is grown in the absence of methanol.

In addition to including host cells containing the vector constructs discussed herein, the present invention also provides primary (primar mammalian) primary (primar) origins.
y) Includes host cells, secondary host cells, and immortalized host cells. These host cells are engineered to delete or replace endogenous genetic material (eg, coding sequences) and / or genetic material (eg, operably linked to a polynucleotide of the invention) Heterologous polynucleotide sequences) and activate, alter, and / or amplify endogenous polynucleotides. For example, techniques known in the art can be used to operably link heterologous regulatory regions (eg, promoters and / or enhancers) and endogenous polynucleotide sequences via homologous recombination, such that Form a new transfer unit (eg, US Pat. No. 5,641,670 issued June 24, 1997; US Pat. No. 5,731 issued March 31, 1998).
No. 3,761; International Publication No. WO 96/29 published on September 26, 1996
411; International Publication No. WO 94/12650 published on August 4, 1994
Koller et al., Proc. Natl. Acad. Sci. USA 86: 8
932-8935 (1989); and Zijlstra et al., Nature 3
42: 435-438 (1989). Each of these disclosures is incorporated by reference in their entirety).

Furthermore, the polypeptides of the present invention can be chemically synthesized using techniques known in the art (eg, Creighton, 1983, Proteins: Stru).
cuts and Molecular Principles, W.C. H.
Freeman & Co. , N.M. Y. And Hunkapiller et al., Na
true, 310: 105-111 (1984)). For example, a polypeptide corresponding to a fragment of the polypeptide sequence of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to: D isomers of normal amino acids, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4
-Aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline , Homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,
Phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids (eg, b-methyl amino acids), Ca-methyl amino acids, Na-methyl amino acids, and common amino acid analogs. In addition, the amino acid is D
(Dextrorotatory) or L (left-handed).

The present invention can be used during or after translation, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, to antibody molecules or other cellular ligands. It includes polypeptides that are differentially modified, such as by binding. Any number of chemical modifications can be performed by known techniques including, but not limited to: specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH ₄ ; acetylation, Formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin;

Additional post-translational modifications included by the present invention include, for example: N-linked or O-linked carbohydrate chains (N-terminal or C-terminal processing), attachment of chemical moieties to the amino acid backbone, N Chemical modification of conjugated or O-linked carbohydrate chains and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The polypeptide can also be modified with a detectable label (eg, an enzyme label, a fluorescent label, an isotope label or an affinity label) to allow detection and isolation of the protein.

The present invention also provides chemically modified derivatives of the polypeptides of the present invention that may provide additional advantages (eg, increased solubility, stability and circulation time of the polypeptide, or decreased immunogenicity) ( See U.S. Pat. No. 4,179,337). The chemical moiety for derivatization can be selected from water-soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, and the like. The polypeptide may be modified at random positions within the molecule or at a predetermined position within the molecule and may include one, two, three or more attached chemical moieties.

The polymer can be any molecular weight polymer and can be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa for ease of handling and manufacturing (the term “about” indicates that some molecules are shown in polyethylene glycol preparations. Heavier than the average molecular weight and some are lighter than the indicated molecular weight)
It is. Desired therapeutic profile (e.g., desired duration of sustained release, effects on biological activity, if any, ease of handling, degree of antigenicity or lack of antigenicity, and for therapeutic proteins or analogs Depending on other known effects of polyethylene glycol, other sizes can be used. For example, polyethylene glycol is about 200, 500, 1000, 1500, 2
000, 2500, 3000, 3500, 4000, 4500, 5000, 55
00, 6000, 6500, 7000, 7500, 8000, 8500, 900
0, 9500, 10,000, 10,500, 11,000, 11,500, 1
2,000, 12,500, 13,000, 13,500, 14,000, 14
, 500, 15,000, 15,500, 16,000, 16,500, 17,
000, 17,500, 18,000, 18,500, 19,000, 19,5
00, 20,000, 25,000, 30,000, 35,000, 40,00
0, 50,000, 55,000, 60,000, 65,000, 70,000
, 75,000, 80,000, 85,000, 90,000, 95,000,
Or it may have an average molecular weight of 100,000 kDa.

As described above, polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in US Pat. No. 5,64.
No. 3,575; Morpurgo et al., Appl. Biochem. Biotec
hnol. 56: 59-72 (1996); Vorobjev et al., Nucleo
side Nucleotides 18: 2745-2750 (1999)
And Calisetti et al., Bioconjug. Chem. 10: 638-
646 (1999), the disclosure of each of which is incorporated herein by reference.

A polyethylene glycol molecule (or other chemical moiety) should be attached to the protein in view of its effect on the functional or antigenic domain of the protein. There are a number of attachment methods available to those skilled in the art (eg, EP 0 401 384 (G-CSF, incorporated herein by reference)).
PEG), Malik et al., Exp. Hematol. 20: 102
8-1035 (1992) (tresyl chloride)
(See also PEGylation of GM-CSF using). For example, polyethylene glycol can be covalently linked by an amino acid residue via a reactive group (eg, a free amino group or a carboxyl group). A reactive group is a group to which an activated polyethylene glycol molecule can bind. Amino acid residues having a free amino group can include lysine residues and N-terminal amino acid residues; amino acid residues having a free carboxyl group include aspartic acid residues, glutamic acid residues and C-terminal amino acid residues Residues may be mentioned. Sulfhydryl residues are also
It can be used as a reactive group for attaching polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol can be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to a protein via a covalent bond to a lysine residue, histidine residue, aspartic acid residue, glutamic acid residue, or cysteine residue.
One or more reaction chemistry is used to select a specific amino acid residue of a protein (eg, lysine, histidine, aspartic acid, glutamic acid or cysteine) or more than one type of amino acid residue of a protein (eg, lysine, Polyethylene glycol can be conjugated to histidine, aspartic acid, glutamic acid, cysteine and combinations thereof.

Proteins chemically modified at the N-terminus may be particularly desirable. Polyethylene glycol is used as an example of the present composition from various polyethylene glycol molecules (
(Depending on molecular weight, branching, etc.), the ratio of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mixture, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminal PEGylated protein obtain. The method for obtaining an N-terminal PEGylated preparation (ie separating this part from other mono-PEGylated moieties if necessary) is by purification of the N-terminal PEGylated material from a population of PEGylated protein molecules. possible. Selective proteins chemically modified with an N-terminal modification can be obtained by reductive alkylation that utilizes the differential reactivity of different types of primary amino groups (lysine vs. N-terminus) available for derivatization in specific proteins. Can be achieved. Under appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group-containing polymer is achieved.

As noted above, pegylation of the proteins of the invention can be achieved by a number of means,
Can be achieved. For example, polyethylene glycol can be connected to the protein either directly or by an intervening linker. A linkerless system for connecting polyethylene glycol to proteins is described by Delgado et al., Crit. Rev. Thera. Drug Ca
rrier Sys. 9: 249-304 (1992); Francis et al., In
tern. J. et al. of Hematol. 68: 1-18 (1998); US Pat. No. 4,002,531; US Pat. No. 5,349,052; WO 95/060.
58; and WO 98/32466. The disclosures of each of these are incorporated herein by reference.

One system for attaching polyethylene glycol directly to an amino acid residue of a protein, without an intervening linker, is tresylated.
MPEG (this is tresyl chloride (ClSO
₂ CH ₂ CF ₃ ) and produced by modifying monomethoxy polyethylene glycol (MPEG). Upon protein reaction with tresylated MPEG, polyethylene glycol
It is directly attached to the amine group of the protein. Accordingly, the present invention includes a protein-polyethylene glycol conjugate produced by reacting a protein of the present invention with a polyethylene glycol molecule having a 2,2,2-trifluoroethanesulfonyl group.

Polyethylene glycol can also be attached to proteins using many different intervening linkers. For example, US Pat. No. 5,612,460 (the entire disclosure of which is incorporated herein by reference) discloses urethane linkers for attaching polyethylene glycol to proteins. A protein-polyethylene glycol conjugate in which polyethylene glycol is attached to the protein by a linker is also MPEG-succinimidyl succinate (succin).
imidyl succinate), MPEG activated by 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichlorophenyl carbonate, MPEG-p-nitrophenol carbonate, and various MPEG-succinate derivatives Can be produced by the reaction of proteins with compounds such as A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is hereby incorporated by reference. PEGylated protein products produced using the reaction chemistry shown herein are included within the scope of the present invention.

The number of polyethylene glycol moieties attached to each protein of the invention (ie, the degree of substitution) can also vary. For example, the PEGylated protein of the present invention is
On average, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 2
Zero or more polyethylene glycol molecules can be linked. Similarly, the average degree of substitution is 1-3, 2-4, 3-5, 4-6 per protein molecule,
5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12
Within such ranges as -14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties. Methods for determining the degree of substitution are described, for example, in Delgado et al., Crit. Rev. Ther
a. Drug Carrier Sys. 9: 249-304 (1992).

The polypeptides of the present invention may be monomers or multimers (ie, dimers,
Trimers, tetramers and higher multimers). The invention therefore relates to monomeric and multimeric polypeptides of the invention, their preparation and compositions comprising them (preferably therapeutic). In certain embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In further embodiments, the multimers of the invention are at least a dimer, at least a trimer, or at least a tetramer.

Multimers included by the present invention can be homomers or heteromers. As used herein, the term homomer is a cD encompassed by a polypeptide corresponding to the amino acid sequence of SEQ ID NO: Y or the deposited cDNA clone.
Polypeptides encoded by NA (including fragments, variants, splice variants, and fusion proteins of the polypeptides described herein)
A multimer that contains only. These homomers can include polypeptides having the same or different amino acid sequences. In certain embodiments, the homomers of the invention are multimers that include only polypeptides having the same amino acid sequence. In another specific embodiment, the homomers of the present invention are multimers comprising polypeptides having different amino acid sequences. In certain embodiments, the multimers of the invention are homodimers (eg, including polypeptides having the same or different amino acid sequences) or homotrimers (eg, including polypeptides having the same and / or different amino acid sequences). . In a further embodiment, the homomeric multimer of the present invention is at least a homodimer, at least a homotrimer or at least a homotetramer.

As used herein, the term heteromer refers to a multimer comprising one or more heterologous polypeptides (ie, polypeptides of different proteins) in addition to the polypeptide of the present invention. In certain embodiments, the multimers of the invention are heterodimers, heterotrimers or heterotetramers. In a further embodiment, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer or at least a heterotetramer.

The multimers of the invention can be the result of hydrophobic, hydrophilic, ionic and / or covalent bonds and / or can be indirectly linked, for example, by liposome formation. Thus, in one embodiment, multimers of the present invention (eg, homodimers or homotrimers) are formed when the polypeptides of the present invention contact each other in solution. In another embodiment, the heteromultimers (
For example, a heterotrimer or heterotetramer) is formed when a polypeptide of the invention is contacted with an antibody against the polypeptide of the invention (including an antibody against a heterologous polypeptide sequence in the fusion protein of the invention) in solution. The In other embodiments, a multimer of the invention is with a polypeptide of the invention and / or
Alternatively, it is formed by a covalent bond between the polypeptides of the present invention. Such a covalent bond is one or more amino acids contained in a polypeptide sequence (eg, contained in a polypeptide described in the sequence listing or encoded by the deposited clone). Residues can be included. In one example, this covalent bond is a bridge between cysteine residues located within the polypeptide sequence that interact in the native (ie, naturally occurring) polypeptide. In another example, this covalent bond is the result of a chemical or recombinant operation. Alternatively, such covalent bonds can include one or more amino acid residues contained in the heterologous polypeptide sequence in the fusion protein of the invention.

In one example, the covalent bond is between heterologous sequences contained in the fusion protein of the invention (see, eg, US Pat. No. 5,478,925). In certain instances, this covalent bond is between heterologous sequences contained in the Fc fusion protein of the invention (as described herein). In another specific example, the covalent bond of the fusion protein of the invention is between heterologous polypeptide sequences derived from another protein (eg, osteoprotegerin) that can form covalently linked multimers (eg, osteoprotegerin). , International Publication No. WO 98/49305, the contents of which are hereby incorporated by reference in their entirety). In another embodiment, two or more polypeptides of the invention are linked through a peptide linker. Examples include the peptide linkers described in US Pat. No. 5,073,627 (incorporated herein by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers can be produced using conventional recombinant DNA techniques.

Another method for preparing a multimeric polypeptide of the invention involves the use of a polypeptide of the invention fused to a leucine zipper polypeptide sequence or an isoleucine zipper polypeptide sequence. Leucine and isoleucine zipper domains are polypeptides that promote multimer formation of the protein in which the domain is found. Leucine zipper originally has some DN
Identified in A-binding protein (Landschulz et al., Science
240: 1759, (1988)) and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of suitable leucine zipper domains for generating the soluble multimeric proteins of the invention are described in PCT application WO 94/10, which is incorporated herein by reference.
308, a leucine zipper domain. A recombinant fusion protein comprising a polypeptide of the invention fused to a peptide sequence that dimerizes or trimerizes in solution is expressed in a suitable host cell, and the resulting soluble multimeric fusion protein is known in the art. It is recovered from the culture supernatant using known techniques.

The trimeric polypeptides of the present invention may provide the advantage of enhanced biological activity. Preferred leucine zipper and isoleucine moieties are those that preferentially form trimers. One example is Hopp, incorporated herein by reference.
A leucine zipper derived from pulmonary surfactant protein D (SPD) as described in e et al. (FEBS Letters 344: 191, (1994)) and US patent application Ser. No. 08 / 446,922. Other peptides derived from naturally occurring trimeric proteins can be used in preparing the trimeric polypeptides of the present invention.

In another example, the proteins of the present invention are linked by interaction between Flag® polypeptide sequences contained in the fusion protein of the present invention comprising Flag® polypeptide sequences. In a further embodiment, the binding of the protein of the invention binds by interaction between the heterologous polypeptide sequence contained in the Flag® fusion protein of the invention and the anti-Flag® antibody.

The multimers of the present invention can be produced using chemical techniques known in the art.
For example, polypeptides desired to be included in the multimers of the invention can be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, eg, US Pat. , 478, 925, which is hereby incorporated by reference in its entirety). In addition, multimers of the present invention are generated using techniques known in the art and include one or more cysteine residues between the cysteine residues located within the sequence of the polypeptide desired to be included in the multimer. Intermolecular crosslinks can be formed (see, eg, US Pat. No. 5,478,925, which is hereby incorporated by reference in its entirety). further,
The polypeptides of the present invention can be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide, and techniques known in the art can be used to modify one or more of these modified It can be applied to produce multimers comprising polypeptides (see, eg, US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety). Furthermore, techniques known in the art can be applied to produce liposomes containing polypeptide components desired to be included in the multimers of the invention (see, eg, US Pat. No. 5,4
78,925, which is hereby incorporated by reference in its entirety).

Alternatively, the multimers of the present invention can be generated using genetic engineering techniques known in the art. In one embodiment, the polypeptides included in the multimers of the invention are recombinantly produced using fusion protein techniques otherwise described herein (eg, as described herein). See US Pat. No. 5,478,925, which is incorporated by reference in its entirety). In certain embodiments, a polynucleotide encoding a homodimer of the invention comprises linking a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide, and then further from the original C-terminus to N-terminus. Produced by linking to a synthetic polynucleotide (which lacks a leader sequence) that encodes the translation product of the polypeptide in the opposite direction to that of (e.g., U.S. Pat. No. 5,478,925). In another embodiment, the recombinant technology described herein, otherwise applied in the art, includes a transmembrane domain (or hydrophobic or signal peptide), and membrane reconstitution technology Produces recombinant polypeptides of the invention that can be incorporated into liposomes (see, eg, US Pat. No. 5,478,925, which is incorporated by reference in its entirety).

(Use of polynucleotide)
Each polynucleotide identified herein can be used in a number of ways as a reagent. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosome identification. Since there are currently few chromosomal marking reagents based on actual sequence data (repetitive polymorphisms), there remains a need to identify new chromosomal markers. Each polynucleotide of the present invention can be used as a chromosomal marker.

Briefly, the sequence can be mapped to the chromosome by preparing PCR primers (preferably 15-25 bp) from the sequence shown in SEQ ID NO: X. Primers can be selected using computer analysis so that the primer does not span more than one predicted exon in the genomic DNA. These primers are then used in the somatic cell hybrid P containing individual human chromosomes.
Used for CR screening. Only those hybrids containing the human gene corresponding to SEQ ID NO: X produce an amplified fragment.

Similarly, somatic cell hybrids place polynucleotides on specific chromosomes P
Provides a quick way of CR mapping. Three or more clones can be assigned per day using a single thermal cycler. Furthermore, polynucleotide sub-localization can be accomplished using a panel of specific chromosomal fragments. Other gene mapping strategies that can be used are in situ hybridization, labeled flow-sorted (labeled
flow sorted) chromosome pre-screening, chromosome-specific cD
Preselection by hybridization to construct an NA library, and computer mapping techniques (eg, Shuler, Trends Biotechnol 16:45, incorporated herein by reference in its entirety.
6-459 (1998)).

The exact chromosomal location of the polynucleotide is also the metaphase chromosome spread (spr
ead) fluorescence in situ hybridization (FISH). This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides between 2,000 and 4,000 bp are preferred. For a review of this technology, see Verma et al., “Human Chromoso.
mes: a Manual of Basic Technologies ", P
See ergamon Press, New York (1988).

For chromosome mapping, polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (to mark multiple sites and / or multiple chromosomes).

The polynucleotides of the present invention comprise radiation hybrid mapping, HAPPY mapping, and long range restriction mapping (lon).
(grange restriction mapping) as well. For a review of these techniques and other techniques known in the art, see, for example, Dear “Genome Mapping: A Practical A
proprach "IRL Press at Oxford Universi
ty Press, London (1997); Aydin, J. et al. Mol. Me
d. 77: 691-694 (1999); Hacia et al., Mol. Psychi
atry 3: 483-492 (1998); Herrick et al., Chromo.
some Res. 7: 409-423 (1999); Hamilton et al., M
methods Cell Biol. 62: 265-280 (2000); and / or Ott, J. et al. Hered. 90: 68-70 (1999), each of which is incorporated herein by reference in its entirety.

Once a polynucleotide has been mapped to a precise chromosomal location, the physical location of the polynucleotide can be used in linkage analysis. Linkage analysis establishes a coherence between the chromosomal location and the presentation of a particular disease. (Disease mapping data can be obtained from, for example, V. McKusick, Mendel.
ian Inheritance in Man (Johns Hopkins
(Available online through the University Welch Medical Library)). Assuming 1 megabase mapping resolution and 1 gene per 20 kb, a cDNA that is accurately located in a chromosomal region associated with disease can be one of 50-500 potential causative genes.

Thus, once co-inheritance is established, differences in polynucleotides and corresponding genes between affected and unaffected individuals can be tested. First, visible structural changes in the chromosome (eg, deletions or translocations) are examined in chromosomal spreads or by PCR. If there is no structural change, the presence of a point mutation is confirmed. A mutation observed in some or all affected individuals but not in normal individuals indicates that the mutation can cause the disease.
However, complete sequencing of polypeptides and corresponding genes from several normal individuals is required to distinguish mutations from polymorphisms. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of genes in affected individuals compared to unaffected individuals can be assessed using the polynucleotides of the invention. Any of these changes (
Altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker.

Thus, the present invention also provides a useful diagnostic method during the diagnosis of a disorder, the method measuring and measuring the expression level of the polynucleotide of the present invention in cells or body fluids from an individual. Comparing the gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in gene expression level relative to the standard is indicative of a disorder.

In yet another embodiment, the invention includes a kit for analyzing a sample for the presence of proliferative and / or cancerous polynucleotides from a test subject. In a general embodiment, the kit comprises at least one polynucleotide probe comprising a nucleotide sequence that specifically hybridizes with a polynucleotide of the invention, and a suitable container. In this particular embodiment, the kit includes two polynucleotide probes that define an internal region of a polynucleotide of the invention, wherein each probe includes a 31 ′ mer end internal to this region. Has two chains. In a further embodiment, the probe may be useful as a primer for polymerase chain reaction amplification.

When the diagnosis of a disorder has already been made by conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or suppressed polynucleotide expression of the present invention can be Experience poor clinical results compared to patients expressing this gene at near levels.

“Measuring the expression level of the polynucleotide of the present invention” means that the level of the polypeptide of the present invention or the level of mRNA encoding this polypeptide is directly measured in the first biological sample (for example, Qualitatively, either by determining or evaluating absolute protein or mRNA levels) or relative (eg, by comparing against polypeptide or mRNA levels in a second biological sample). It is intended to be measured or evaluated automatically or quantitatively Preferably, the polypeptide level or mRNA level in the first biological sample is measured or evaluated and compared against a standard polypeptide level or mRNA level, the standard from an individual without a disorder. Determined by averaging the levels obtained from the resulting second biological sample or from a population of individuals who do not have the disorder. As recognized in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained from an individual, body fluid, cell line, tissue culture or other source that contains a polypeptide or mRNA of the invention. As indicated, a biological sample can express a body fluid (eg, semen, lymph, serum, plasma, urine, synovial fluid and cerebrospinal fluid) containing a polypeptide of the invention, and a polypeptide of the invention. Includes other tissue sources found.
Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.
If the biological sample contains mRNA, tissue biopsy is the preferred source.

The methods provided above can preferably be applied to diagnostic methods and / or kits in which the polynucleotide and / or polypeptide is bound to a solid support. In one exemplary method, the support is described in US Pat. Nos. 5,837,832, 5,874,219 and 5,856,174, “
It can be a “gene chip” or a “biological chip”. Furthermore, such a gene chip to which a polynucleotide of the invention is bound can be used to identify polymorphisms between the polynucleotide sequence and a polynucleotide isolated from a test subject. Such polymorphic findings (ie, their location as well as their presence) are useful in identifying disease loci for many disorders, including cancerous diseases and conditions. Such methods are described in US Pat. Nos. 5,858,659 and 5,856,104. The above referenced US patents are hereby incorporated by reference in their entirety.

The invention encompasses polynucleotides of the invention that are chemically synthesized (ie, reproduced as peptide nucleic acids (PNA)) or synthesized according to other methods known in the art. The use of PNA serves as a preferred form when the polynucleotide is incorporated into a solid support, ie a gene chip. For the purposes of the present invention, peptide nucleic acids (PNA) are polyamide-type DNA analogues and monomeric units for adenine, guanine, thymine and cytosine are commercially available (Perceptitis Biosystems). Certain components of DNA (eg, phosphorous, phosphorous oxide or deoxyribose derivatives) are PN
Not present in A. P. E. Nielsen, M.M. Egholm, R.A. H. Be
rg and O.I. Buchardt, Science 254, 1497 (199
1); Egholm, O .; Buchardt, L.M. Christe
nsen, C.I. Behrens, S.M. M.M. Freier, D.M. A. Driver
, R. H. Berg, S.M. K. Kim, B.M. Norden and P.M. E. Nie
As disclosed by lsen, Nature 365, 666 (1993), PNA binds specifically and tightly to complementary DNA strands and is not degraded by nucleases. In fact, PNA binds to DNA more strongly than DNA itself binds. This is probably due to the absence of electrostatic repulsion between the two chains and the polyamide backbone is also more flexible. As a result, P
NA / DNA duplexes bind under a wider range of stringency conditions than DNA / DNA duplexes, making it easier to perform multi-stranded hybridization.
Due to strong binding, smaller probes can be used than with DNA.
Furthermore, perhaps a single base mismatch can be determined using PNA / DNA hybridization. This is because a single mismatch in the 15-mer of PNA / DNA is between 4 ° C. and 16 ° C. for the 15-mer duplex of DNA / DNA, whereas the melting point (T.sub.m) is 8-20 This is because the temperature is lowered.
Also, the absence of charged groups in PNA means that hybridization can be performed at low ionic strength and reduces possible interference with salts during analysis.

The present invention is useful for the detection of cancer in mammals. In particular, the present invention
Useful during the diagnosis of pathological cell proliferation neoplasia, including but not limited to: acute myeloid leukemia (acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, Acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia,
And acute undifferentiated leukemia); and chronic myelogenous leukemia (including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc.). Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred is human.

Pathological cell proliferative diseases, disorders and / or conditions are often associated with inappropriate activation of proto-oncogenes (Gelmann, EP et al., “The Eti
olology of Accurate Leukemia: Molecular Ge
nets and Viral Oncology ", Neoplasti
c Diseases of the Blood, Volume 1, Wiernik,
P. H. Et al., 161-182 (1985)). Neoplasia currently involves qualitative changes in normal cellular gene products, either by insertion of viral sequences into the chromosome, chromosomal translocation of the gene to a more actively transcribed region, or some other mechanism, or of gene expression It is believed to result from quantitative modification (Gelmann et al., Supra). Certain gene mutations or altered expression appear to be involved in several leukemia etiologies, among other tissues and other cell types (Gelmann et al., Supra).
Indeed, the human counterparts of oncogenes involved in some animal neoplasia are amplified or translocated in some cases of human leukemia and carcinoma (Gelman
n et al., supra). For example, c-myc expression is expressed in non-lymphocytic leukemia cell line HL-6.
Highly amplified at zero. When HL-60 cells are chemically induced to stop amplification, the level of c-myc is found to be down-regulated (International Publication No. WO 91/15580). However, c-myc or c-
Exposure of HL-60 cells to a DNA construct that is complementary to the 5 ′ end of myb
-It has been shown to block translation of the corresponding mRNA that down-regulates the expression of myc protein or c-myb protein, resulting in arrest of cell proliferation and differentiation of the treated cells (International Publication No. WO 91/15580) ;
Wickstrom et al., Proc. Natl. Acad. Sci. 85: 102
8 (1988); Anfossi et al., Proc. Natl. Acad. Sci.
86: 3379 (1989)). However, given the number of cells and cell types of various origins known to exhibit a proliferative phenotype, those skilled in the art will appreciate the utility of the present invention for proliferative diseases and disorders of hematopoietic cells and tissues And / or recognize that it is not limited to treating the condition.

In addition to the foregoing, the polynucleotide may comprise triple helix formation or antisense DN.
It can be used to control gene expression through A or RNA. Antisense technology is described, for example, in Okano, J. et al. Neurochem. 56: 560 (1
991), “Oligodeoxynucleotides as Antis.
ense Inhibitors of Gene Expression ",
Considered in CRC Press, Boca Raton, FL (1988). Triple helix formation is described, for example, by Lee et al., Nucleic Acids Re.
search 6: 3073 (1979); Cooney et al., Science.
241: 456 (1988); and Dervan et al., Science 251.
: 1360 (1991). Both methods rely on binding of the polynucleotide to complementary DNA or complementary RNA. For these techniques, preferred polynucleotides are usually oligonucleotides of 20-40 bases in length, and gene regions involved in transcription (triple helix-Lee et al., Nucl. A).
cids Res. 6: 3073 (1979); Cooney et al., Science
e 241: 456 (1988); and Dervan et al., Science 2
51: 1360 (1991)) or mRNA itself (antisense-Okano, J. Neurochem. 56: 560 (1991); Olig
odeoxy-nucleotides as Antisense Inhi
bits of Gene Expression, CRC Press,
Boca Raton, FL (1988)). Triple helix formation optimally results in blockage of RNA transcription from DNA, while antisense RNA hybridization prevents translation of mRNA molecules into polypeptides. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an attempt to treat or prevent disease. .

The polynucleotides of the invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism with a defective gene in an attempt to correct the genetic defect. The polynucleotide disclosed in the present invention is:
It provides a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that did not exist in the host genome, thereby creating a new trait in the host cell.

Polynucleotides are also useful for identifying individuals from small biological samples. For example, the US military is considering the use of restriction fragment length polymorphism (RFLP) to identify its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes and probed for Southern blots to generate unique bands to identify personnel. This method does not suffer from the current limitations of “Dog tags” (which can make positive identification difficult by being lost, exchanged, or stolen). The polynucleotides of the present invention can be used as additional DNA markers for RFLP.

The polynucleotides of the invention can also be used as an alternative to RFLP by determining the actual base-by-base DNA sequence of a selected portion of an individual's genome. These sequences can be used to prepare PCR primers to amplify and isolate such selected DNA, and then the selected DNA can be sequenced. Because each individual has a unique set of DNA sequences, this technique can be used to identify individuals. Once a unique ID database is established for an individual, positive identification of the individual can be made from a very small tissue sample, whether the individual is alive or dead.

Forensic biology also benefits from using DNA-based identification techniques as disclosed herein. Tissue (eg, hair or skin) or body fluid (eg, blood, saliva, semen, synovial fluid, amniotic fluid, milk, lymph, pulmonary sputum)
DNA sequences obtained from very small biological samples (such as utum) or surfactant, urine, fecal material, etc.) can be amplified using PCR.
In one prior art, gene sequences amplified from polymorphic loci such as DQa class II HLA genes are used in forensic biology to identify individuals. (Errich, H., PCR Technology, Freem
an and Co. (1992)). Once these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes. This yields an identification set of bands for Southern blots probed with DNA corresponding to the DQa class II HLA gene. Similarly, the polynucleotides of the invention can be used as polymorphic markers for forensic purposes.

There is also a need for a reagent that can identify the source of a particular tissue. For example, this need arises in forensic medicine when tissue of unknown origin is provided. Suitable reagents may include, for example, DNA probes or primers specific for a particular tissue prepared from the sequences of the present invention. Such a panel of reagents can identify tissues by species and / or organ type. In a similar manner, these reagents can be used to screen tissue cultures for contaminants.

At least, the polynucleotides of the present invention serve as molecular weight markers in Southern gels, as diagnostic probes for the presence of specific mRNAs in specific cell types, to “subtract” known sequences in the process of discovering new polynucleotides. Used as a probe for DNA, to select and create oligomers for attachment to "gene chips" or other supports, to elicit anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response Can be done.

(Use of polypeptides)
Each polypeptide identified herein can be used in a number of ways. The following description should be considered exemplary and utilizes known techniques.

The polypeptides of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissues can be studied using classical immunohistochemistry (Jalka
enn, M.M. Et al. Cell. Biol. 101: 976-985 (1985
Jalkanen, M .; Et al. Cell. Biol. 105: 3087-
3096 (1987)). Other antibody-based methods that are useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include, for example, enzyme labels such as glucose oxidase, as well as iodine (125I, 121I), carbon (14
C), sulfur (35S), tritium (3H), indium (112In), and radioisotopes such as technetium (99mTc), and fluorescent labels such as fluorescein and rhodamine, and biotin.

In addition to assaying secreted protein levels in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for imaging proteins in vivo include those detectable by X-ray radiography, NMR or ESR. For x-ray radiography, suitable labels include radioactive isotopes such as barium or cesium that emit detectable radiation but are not clearly harmful to the subject. NM
Suitable markers for R and ESR include those having a detectable characteristic spin, such as deuterium, which can be incorporated into antibodies by labeling nutrients for the associated hybridoma. .

A protein-specific antibody or antibody labeled with a suitable detectable imaging moiety such as a radioisotope (eg 131I, 112In, 99mTc), radiopaque material, or material detectable by nuclear magnetic resonance The fragment is introduced into the mammal (eg, parenterally, subcutaneously or intraperitoneally). It will be appreciated in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety needed to produce a diagnostic image. In the case of a radioisotope moiety,
For human subjects, the amount of radioactivity injected is usually about 5-20 of 99mTc.
The range of millicuries. The labeled antibody or antibody fragment is then preferentially accumulated at the location of the cell containing the particular protein. In vivo tumor imaging is described in S.H. W. Burchiel et al., “Immunopharmacokine.
tics of Radiolabeled Antibodies and
Their Fragments. (Tumor Imaging: The
The first of Radiochemical Detection of Cancer
Chapter 3, S.M. W. Burchiel and B.M. A. Edited by Rhodes, Masson
Publishing Inc. (1982)).

Accordingly, the present invention provides a method for diagnosing a disorder, the method comprising: (a) assaying the expression of a polypeptide of the present invention in a cell or body fluid of an individual; (b) A step of comparing to the gene expression level, whereby an increase or decrease in the gene expression level of the assayed polypeptide compared to the standard expression level is indicative of the disorder. With regard to cancer, can the presence of a relatively large amount of transcripts in biopsy tissue from an individual indicate a predisposition to the development of the disease,
Or it can provide a means to detect this disease before the appearance of actual clinical symptoms.
This type of more definitive diagnosis may allow health professionals to use preventive measures or aggressive treatment earlier thereby preventing the onset or further progression of cancer.

In addition, the polypeptides of the invention can be used to treat, prevent and / or diagnose diseases. For example, the patient may replace the absence or decrease in level of a polypeptide (eg, insulin), a different polypeptide (eg, hemoglobin B
Supplementing the absence or reduction of the level of hemoglobin S, SOD, catalase, DNA repair protein), inhibiting the activity of a polypeptide (eg, an oncogene or tumor suppressor), the activity of a polypeptide (eg, Activating by binding to the receptor, reducing the activity of the membrane-bound receptor by competing with the membrane-bound receptor for a free ligand (eg, a soluble TNF receptor used in reducing inflammation), or In addressing the desired response (eg, inhibiting vascular growth, enhancing the immune response to proliferating cells or tissues), the polypeptides of the invention can be administered.

Similarly, antibodies against the polypeptides of the invention can also be used to treat, prevent and / or diagnose diseases. For example, administration of an antibody against a polypeptide of the invention can bind to the polypeptide and reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, for example, by binding to a membrane-bound polypeptide (receptor).

At least the polypeptide of the present invention can be obtained by SDS-P using methods well known to those skilled in the art.
It can be used as a molecular weight marker for AGE gels or molecular sieve gel filtration columns. Polypeptides can also be used to raise antibodies, which are then used to measure protein expression from recombinant cells as a method of assessing host cell transformation. In addition, the polypeptides of the invention can be used to test the following biological activities:

(Gene therapy method)
Another aspect of the invention relates to gene therapy methods for treating or preventing disorders, diseases, and conditions. This gene therapy method uses nucleic acids (DNA, RNA, and antisense DNA or RNA to achieve expression of the polypeptides of the invention.
) Relates to the introduction of sequences into animals. This method requires a polynucleotide encoding a polypeptide of the present invention operably linked to a promoter and any other genetic elements necessary for expression of this polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, eg, WO 90/11092, incorporated herein by reference.

Thus, for example, a patient-derived cell is a polynucleotide (DNA or R) comprising a promoter operably linked to a polynucleotide of the invention ex vivo.
NA) and the engineered cells are then provided to the patient to be treated with the polypeptide. Such methods are well known in the art.
See, for example, Belldegrun et al. Natl. Cancer Inst. ,
85: 207-216 (1993); Ferrantini et al., Cancer
Research, 53: 107-1112 (1993); Ferrantin
i et al. Immunology 153: 4604-4615 (1994);
Kaido, T .; Et al., Int. J. et al. Cancer 60: 221-229 (19
95); Ogura et al., Cancer Research 50: 5102-5.
106 (1990); Santodonato et al., Human Gene Th
erapy 7: 1-10 (1996); Santodonato et al., Gene.
See Therapy 4: 1246-1255 (1997); and Zhang et al., Cancer Gene Therapy 3: 31-38 (1996). These documents are incorporated herein by reference. In one embodiment, the engineered cell is an arterial cell. The arterial cells can be reintroduced into the patient by direct injection into the artery, tissue surrounding the artery, or by catheter injection.

As discussed in more detail below, the polynucleotide construct can be used in any method that delivers injectable material to animal cells (eg, tissue (heart, muscle, skin,
Injection) into the interstitial space of the lung, liver, etc.). The polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

In one embodiment, the polynucleotides of the invention are delivered as naked polynucleotides. The term “naked” polynucleotide, DNA or RNA is any delivery vehicle that acts to assist, facilitate or facilitate cell entry (
It also includes sequences that do not include viral sequences, viral particles, liposome formulations, lipofectins, or precipitants. However, the polynucleotides of the present invention can also be delivered in liposome formulations, and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in US Pat. Nos. 5,593,972 and 5,589, which are incorporated herein by reference.
, 466 and 5,580,859.

The polynucleotide vector constructs of the invention used in gene therapy methods are preferably not integrated into the host genome and do not contain sequences that allow replication.
Is a construct. Suitable vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; Phar
pSVK3, pBPV, pMSG and pSVL available from macia; and pEF1 / V5, pcDNA3.1 available from Invitrogen
And pRc / CMV2. Other suitable vectors will be readily apparent to those skilled in the art.

Any strong promoter known to those skilled in the art can be used to drive expression of the polynucleotide sequences of the invention. Suitable promoters include adenovirus promoters (eg, adenovirus major late promoter); or heterologous promoters (eg, cytomegalovirus (CMV) promoter);
RS virus (RSV) promoter; inducible promoter (eg, MMT promoter, metallothionein promoter); heat shock promoter; albumin promoter; ApoAI promoter; human globin promoter; viral thymidine kinase promoter (eg, herpes simplex thymidine kinase promoter); retrovirus LTR; b-actin promoter; and human growth hormone promoter. This promoter may also be a native promoter for the polynucleotide of the present invention.

Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transient nature of polynucleotide synthesis in those cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for a period of up to 6 months.

The polynucleotide construct of the present invention is used for tissue in animals (muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testis. , Ovary, uterus, rectum, nervous system, eye, gland, and connective tissue)
In the interstitial space. This interstitial space of tissue is between reticulofibers of organ tissue,
Liquid mucopolysaccharide matrix between cells, elastic fibers in the walls of blood vessels or chambers, collagen fibers of fibrous tissue, or connective tissue sheath muscle cells
including the same substrate within the ishes enhesing muscle cell) or in the hiatus of the bone. This is likewise the space occupied by circulating plasma and lymph fluid in the lymph channels. Delivery of muscle tissue into the interstitial space
Preferred for reasons discussed below. The polynucleotide construct of the present invention comprises:
It can be conveniently delivered by injection into tissues containing these cells. The polynucleotide constructs of the present invention are preferably delivered to and expressed in differentiated, persistent, non-dividing cells, but delivery and expression is non-differentiated cells or cells that are not fully differentiated ( For example, it can be achieved in blood stem cells or dermal fibroblasts). Muscle cells in vivo take up the polynucleotide,
And it is particularly qualified for its ability to develop.

For naked nucleic acid sequence injections, an effective dosage of DNA or RNA is about 0.05.
It is in the range of mg / kg body weight to about 50 mg / kg body weight. Preferably, the dosage is from about 0.005 mg / kg to about 20 mg / kg, and more preferably from about 0.05 mg / kg to about 5 mg / kg. Of course, as those skilled in the art will appreciate, this dosage will vary depending on the tissue site of the injection. Appropriate and effective dosages of the nucleic acid sequences can be readily determined by one skilled in the art and can depend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes can also be used, including, for example, inhalation of aerosol formulations for delivery to, for example, lung or bronchial tissue, throat or nasal mucosa. Furthermore, naked DNA constructs can be delivered to the artery by the catheter used in this procedure during angioplasty.

Naked polynucleotides are delivered by any method known in the art including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter injection, and so-called “gene guns”. The These delivery methods are known in the art.

This construct can also be delivered using delivery vehicles such as viral sequences, viral particles, liposomal formulations, lipofectin, precipitants, and the like. Such delivery methods are known in the art.

In certain embodiments, the polynucleotide constructs of the invention are complexed in a liposome preparation. Liposome preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because they can form a strong charge complex between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes are in functional form in plasmid DNA (Felner et al., Proc. Natl. Acad. S, incorporated herein by reference.
ci. USA, 84: 7413-7416 (1987)); mRNA (Malone et al., Proc. Natl. Acad.
Sci. USA, 86: 6077-6081 (1989)); and purified transcription factors (Debs et al., J. Biol. C, incorporated herein by reference).
hem. 265: 10189-10192 (1990)) has been shown to mediate intracellular delivery.

Cationic liposomes are readily available. For example, N [1-2,3-dioleyloxy) propyl] -N, N, N-triethylammonium (DOTM)
A) Liposomes are particularly useful and are registered under the registered trademark Lipofectin under GIBCO BRL, Grand Island, N.M. Y. (Felgner et al., Proc. Natl. Acad. Sc, incorporated herein by reference.
i. USA, 84: 7413-7416 (1987), also available). Other commercially available liposomes include transfections (t
random) (DDAB / DOPE) and DOTAP / DOPE
(Boehringer).

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. DOTAP (1,2-bis (oleoyloxy)-
For a description of the synthesis of 3- (trimethylammonio) propane) liposomes, see, eg, PCT Publication No. WO 90/11092 (incorporated herein by reference). The preparation of DOTMA liposomes has been described in the literature, see, eg, Felgner et al., Proc. Na
tl. Acad. Sci. USA, 84: 7413-7417. Similar methods can be used to prepare liposomes from other cationic lipid substances.

Similarly, anionic liposomes and neutral liposomes are, for example, Avant
i Polar Lipids (Birmingham, Ala.) are readily available or can be easily prepared using readily available materials.
Among such substances are phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), among others.
), Dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE). These substances are also
Can be mixed with DOTMA starting material and DOTAP starting material in appropriate proportions. Methods for producing liposomes using these materials are well known in the art.

For example, commercially dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) are used in various combinations, with or without the addition of cholesterol. Conventional liposomes can be produced. Therefore,
For example, DOPG / DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonicated vial. The sample is placed under a vacuum pump overnight and hydrated with deionized water the next day. The sample was then placed in a capped vial using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at maximum setting while the bath was circulating at 15EC. Sonicate for 2 hours at Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles, or single-sized vesicles of different sizes can be obtained by extruding through a nuclear pore membrane. Can be generated. Other methods are known and available to those skilled in the art.

The liposome may include multilamellar liposomes (MLV), small unilamellar liposomes (SUV) or large unilamellar liposomes (LUV), with SUV being preferred. Various liposome-nucleic acid complexes are prepared using methods well known in the art. For example, Straubinge, incorporated herein by reference.
r et al., Methods of Immunology, 101: 512-527.
(1983). For example, an MLV containing nucleic acid can be prepared by depositing a thin film of phospholipid on the wall of a glass tube and then hydrating with a solution of the substance to be encapsulated. SUVs are prepared by long-term sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The substance to be encapsulated is added to the preformed suspension of MLV and then sonicated. When using liposomes containing cationic lipids, dry lipid membranes are treated with sterile water or 10
Resuspend in a suitable solution, such as an isotonic buffer solution such as mM Tris / NaCl, sonicate and then mix the preformed liposomes directly with DNA. Due to the binding of positively charged liposomes to cationic DNA, liposomes and DNA form very stable complexes. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods well known in the art. Commonly used methods include Ca ²⁺ -EDTA chelation (Pap
ahadjopoulos et al., Biochim. Biophys. Acta, 3
94: 483 (1975); Wilson et al., Cell, 17:77 (1979).
)); Ether injection (Deamer et al., Biochim. Biophys. Ac)
ta, 443: 629 (1976); Ostro et al., Biochem. Biop
hys. Res. Commum. 76: 836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA, 76: 3348 (1979).
)); Surfactant dialysis (Enoch et al., Proc. Natl. Acad. Sci)
. USA, 76: 145 (1979)); and reverse phase evaporation (REV).
(Fraley et al., J. Biol. Chem. 255: 10431 (198).
0); Szoka et al., Proc. Natl. Acad. Sci. USA, 75:
145 (1978); Schaefer-Ridder et al., Science, 2
15: 166 (1982)), which are hereby incorporated by reference.

Generally, the ratio of DNA to liposome is from about 10: 1 to about 1:10. Preferably, the ratio is from about 5: 1 to about 1: 5. More preferably, the ratio is from about 3: 1 to about 1: 3. Even more preferably, the ratio is about 1: 1.

US Pat. No. 5,676,954 (incorporated herein by reference) reports the injection of genetic material complexed with a cationic liposome carrier into mice. U.S. Pat.Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466
No. 5,693,622, No. 5,580,859, No. 5,703,
055 and International Publication No. WO 94/9469, which are incorporated herein by reference, provide cationic lipids for use in transfecting DNA into cells and mammals. US Pat. No. 5,589,466,
No. 5,693,622, No. 5,580,859, No. 5,703,05
No. 5 and International Publication No. WO 94/9469, which are incorporated herein by reference, provide methods for delivering DNA-cationic lipid complexes to mammals.

In certain embodiments, an RN comprising a sequence encoding a polypeptide of the invention
Manipulate cells ex vivo or in vivo using retroviral particles containing A. Retroviruses from which retroviral plasmid vectors can be derived include Moloney murine leukemia virus, spleen necrosis virus, rous sarcoma virus,
Harvey sarcoma virus, chicken leukemia virus, gibbon leukemia virus,
These include, but are not limited to, human immunodeficiency virus, myeloproliferative sarcoma virus, and breast cancer virus.

This retroviral plasmid vector is used to transduce packaging cell lines to form producer cell lines. Examples of packaging cell lines that can be transfected include Miller, Human Gene Therapy, 1: 5-14 (199, which is hereby incorporated by reference in its entirety.
0) PE501, PA317, R-2, R-AM, PA1
2, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP +
These include, but are not limited to, E-86, GP + envAm12 and DAN cell lines. This vector can transduce packaging cells by any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO ₄ precipitation. In one alternative, the retroviral plasmid vector can be encapsulated in liposomes or conjugated to lipids and then administered to the host.

This producer cell line produces infectious retroviral vector particles comprising a polynucleotide encoding a polypeptide of the invention. Such retroviral vector particles can then be used to transduce eukaryotic cells either in vitro or in vivo. This transduced eukaryotic cell expresses a polypeptide of the invention.

In certain other embodiments, the polynucleotides of the invention contained in adenoviral vectors are used to manipulate cells ex vivo or in vivo. An adenovirus can be engineered such that it encodes and expresses a polypeptide of the invention and at the same time is inactivated with respect to its ability to replicate in the normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thus reducing concerns about insertional mutagenesis. In addition, adenoviruses have been used for many years with excellent safety aspects as live gut vaccines (Schwartzet et al., Am. Re
v. Respir. Dis. 109: 233-238 (1974)). Finally, adenovirus-mediated gene transfer has been demonstrated in many examples, including transfer of α-1-antitrypsin and CFTR to the lungs of cotton rats (Ro
senfeld et al., Science, 252: 431-434 (1991); R
osenfeld et al., Cell, 68: 143-155 (1992)). Furthermore, extensive studies attempting to establish adenovirus as a causative agent in human cancer were uniformly negative (Green et al. Proc. Natl. Acad. S).
ci. USA, 76: 6606 (1979)).

Suitable adenoviral vectors useful in the present invention are, for example, Koz
arsky and Wilson, Curr. Opin. Genet. Devel
. 3: 499-503 (1993); Rosenfeld et al., Cell, 68.
: 143-155 (1992); Engelhardt et al., Human Gen.
et. Ther. 4: 759-769 (1993); Yang et al., Nature.
e Genet, 7: 362-369 (1994); Wilson et al., Natu.
re, 365: 691-692 (1993); and US Pat. No. 5,652,2.
24, which are incorporated herein by reference. For example, the adenoviral vector Ad2 is useful and can be propagated in human 293 cells. These cells contain the E1 region of adenovirus and constitutively express Ela and Elb, which complement the defective adenovirus by providing the product of the gene that is missing from this vector. . In addition to Ad2, a variety of other adenoviruses (eg, Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenovirus used in the present invention is replication defective. Replication-deficient adenoviruses require the help of helper viruses and / or packaging cell lines to form infectious particles. The resulting virus is capable of infecting cells and can express the polynucleotide of interest operably linked to a promoter, but cannot replicate in most cells. The replication-deficient adenovirus can be deleted in one or more of the following genes: E1a, E1b, E3, E4, E2a or all or part of L1 to L5.

In certain other embodiments, adeno-associated virus (AAV) is used to manipulate the cells ex vivo or in vivo. AAV is a naturally occurring defective virus that requires a helper virus to generate infectious particles (
Muzyczka, Curr. Topics in Microbiol. Im
munol. 158: 97 (1992)). AAV is also one of the few viruses that can incorporate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and integrated,
Space for exogenous DNA is limited to about 4.5 kb. Methods for generating and using such AAV are known in the art. For example, US Pat. No. 5,139.
941, No. 5,173,414, No. 5,354,678, No. 5,
436,146, 5,474,935, 5,478,745 and 5,589,377.

For example, suitable AAV vectors for use in the present invention contain all the sequences necessary for DNA replication, encapsidation, and host cell integration. Sam
Brook et al., Molecular Cloning: A Laborator
y Manual, Cold Spring Harbor Press (19
Using standard cloning methods, such as the method found in 89), a polynucleotide construct comprising a polynucleotide of the invention is inserted into this AAV vector. The recombinant AAV vector is then transfected into packaging cells infected with helper virus using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, and the like. Suitable helper viruses include adenovirus, cytomegalovirus, vaccinia virus, or herpes virus. Once the packaging cells are transfected and infected, they produce infectious AAV virions containing the polynucleotide constructs of the invention. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cell contains a polynucleotide construct integrated into its genome and expresses the desired gene product.

Another method of gene therapy is homologous recombination (eg, US Pat. No. 5,641,67).
No. 0, issued on June 24, 1997; International Publication No. WO 96/29411, 199
Published on Sep. 26, 6; International Publication No. WO94 / 12650, published on Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86
: 8932-8935 (1989); and Zijlstra et al., Nature.
342: 435-438 (1989)), operably linking the heterologous regulatory region and the endogenous polynucleotide sequence (eg, the sequence encoding the polypeptide sequence of interest). Including. This method involves the activation of a gene that is present in the target cell but is usually not expressed in that cell or expressed at a lower level than desired.

Polynucleotide constructs are made using standard techniques known in the art. This construct includes a promoter with a targeting sequence adjacent to the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to the endogenous sequence, allowing homologous recombination between the promoter-targeting sequence and the endogenous sequence. The targeting sequence is present sufficiently close to the 5 ′ end of the desired endogenous polynucleotide sequence, and thus, upon homologous recombination, the promoter is operably linked to the endogenous sequence.

This promoter and targeting sequence can be amplified using PCR. Preferably, the amplified promoter contains separate restriction enzyme sites at the 5 'and 3' ends. Preferably, the 3 ′ end of the first targeting sequence comprises the same restriction enzyme site as the 5 ′ end of the amplified promoter, and the 5 ′ end of the second targeting sequence is 3 of the amplified promoter. 'Contains the same restriction sites as the ends. The amplified promoter and targeting sequence are digested and ligated together.

Liposomes, viral sequences, viral particles, whole viruses, lipofections, as naked polynucleotides or as described in more detail above,
This promoter-targeting sequence construct is delivered to the cell either in conjunction with a transfection facilitating agent such as a precipitant. Direct needle injection, intravenous injection,
The P promoter-targeting sequence can be delivered by any method including local administration, catheter injection, particle accelerator, and the like. This method is described in more detail below.

The promoter-targeting sequence construct is taken up by the cell. Homologous recombination occurs between this construct and the endogenous sequence, so that the endogenous sequence is placed under the control of this promoter. This promoter then drives the expression of the endogenous sequence.

A polynucleotide encoding a polypeptide of the invention can be administered together with other polynucleotides encoding other angiogenic proteins. Angiogenic proteins include acidic and basic fibroblast growth factors, VEGF-1, V
EGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor α
And β, platelet derived endothelial cell growth factor, platelet derived growth factor, tumor necrosis factor α, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte / macrophage colony stimulating factor, and one Examples include, but are not limited to, nitric oxide synthase.

Preferably, the polynucleotide encoding the polypeptide of the present invention comprises a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is located in the coding region of the polynucleotide that is expressed toward or at the 5 'end of the polynucleotide coding region. This signal sequence can be homologous or heterologous to the polynucleotide of interest and can be homologous or heterologous to the transfected cell. In addition, the signal sequence can be chemically synthesized using methods known in the art.

Any dosage form of any of the above polynucleotide constructs can be used as long as the dosage form expresses one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter injection, biolistic syringes, particle accelerators (ie, “gene guns”), gelfoam sponge depots, other commercial depots (de
pot) materials, osmotic pumps (eg, Alza mini pumps), solid (tablets or pills) pharmaceutical formulations for oral or suppository, and decanting or topical application during surgery. For example, direct injection of calcium phosphate-precipitated naked plasmid into rat liver and rat spleen, or direct protein-coated plasmid injection into portal vein resulted in gene expression of foreign genes in rat liver (Ka
neda et al., Science, 243: 375 (1989)).

The preferred method of local administration is by direct injection. Preferably, the recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection within the arterial region or locally administered within the arterial region. Topical administration of a composition within the arterial area refers to injecting the composition into the artery several centimeters, preferably several millimeters.

Another method of topical administration is to contact the polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can experience surgery and the polynucleotide construct can be coated on the tissue surface inside the wound, or the construct can be injected into a tissue region inside the wound.

A therapeutic composition useful for systemic administration comprises a recombinant molecule of the invention complexed with a targeted delivery vehicle of the invention. Suitable delivery vehicles for use in systemic administration include liposomes containing a ligand that targets the vehicle to a specific site.

Preferred methods of systemic administration include intravenous injection, aerosol, oral and transdermal (topical) delivery. Intravenous injection can be performed using standard methods in the art. Aerosol delivery can also be performed using standard methods in the art (eg, Striving, incorporated herein by reference).
Et al., Proc. Natl. Acad. Sci. USA, 189: 11277-1
1281 (1992)). Oral delivery can be performed by complexing the polynucleotide construct of the present invention to a carrier capable of resisting degradation by digestive enzymes in the intestine of the animal. Examples of such carriers include
Examples include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a lipophilic reagent that can pass into the skin (eg, DMSO) and a polynucleotide construct of the invention.

Determining an effective amount of a substance to be delivered includes, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the exact condition requiring treatment and its severity, and the route of administration Can depend on a number of factors. The frequency of treatment is
It depends on many factors, such as the amount of polynucleotide construct administered per dose and the health and medical history of the subject. The exact amount, number of doses and timing of dosing will be determined by the attending physician or attending veterinarian. The therapeutic composition of the present invention comprises:
It can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits, sheep, cows, horses and pigs, especially humans.

(Biological activity)
The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides show activity in a particular assay, these molecules are likely to be involved in diseases associated with their biological activity. Thus, the polynucleotide or polypeptide, or agonist or antagonist can be used to treat a related disease.

(Immunoactivity)
The polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the present invention, for example, activate or inhibit immune cell proliferation, differentiation, or mobilization (chemotaxis), thereby causing diseases or disorders of the immune system. And / or conditions may be useful for treating, preventing, and / or diagnosing. Immune cells develop through a process called hematopoiesis and produce myeloid cells (platelets, erythrocytes, neutrophils and macrophages) and lymphoid cells (B lymphocytes and T lymphocytes) from pluripotent stem cells. The pathogenesis of these immune diseases, disorders and / or conditions can be genetic, somatic (eg, cancer or some autoimmune disorders), acquired (eg, due to chemotherapy or toxins) or infectious. possible. Furthermore, the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the present invention can be used as markers or detectors for diseases or disorders of a particular immune system.

The polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the present invention may be used to treat hematopoietic cell diseases, disorders and / or conditions,
It can be useful in prevention and / or diagnosis. The polynucleotide of the present invention,
Polypeptides, antibodies, and / or agonists or antagonists include pluripotent stem cells in an attempt to treat or prevent diseases, disorders and / or conditions associated with the reduction of certain (or many) types of hematopoietic cells. It can be used to increase the differentiation and proliferation of hematopoietic cells. Examples of immunodeficiency syndromes include blood protein diseases, disorders and / or conditions (eg, agammaglobulinemia, hypogammaglobulinemia), telangiectasia ataxia, unclassifiable immune deficiency,
Di George syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion syndrome, lymphopenia, phagocytic bactericidal dysfunction, severe combined immunodeficiency (SCID)
, Viscott-Oldrich disorder, anemia, thrombocytopenia, or hemoglobinuria, but are not limited thereto.

Furthermore, the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the present invention may also be used to modulate hemostatic activity (stop bleeding) or thrombolytic activity (clot formation). For example, by increasing the hemostatic activity or thrombolytic activity, the polynucleotides or polypeptides, and / or agonists or antagonists of the present invention may be used to treat blood coagulation diseases, disorders and / or conditions (eg, fibrinogens). Blood, blood platelet diseases, disorders and / or conditions (eg, thrombocytopenia), or wounds resulting from trauma, surgery or other causes may be treated or prevented. Or
The polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the present invention that can reduce hemostatic or thrombolytic activity can be used to inhibit or lyse blood clots. These molecules may be important in the treatment or prevention of a heart attack (infarction), stroke or scar.

In the treatment, prevention and / or diagnosis of autoimmune disorders, the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the present invention may also be useful. Many autoimmune disorders result from inappropriate self-recognition as foreign substances by immune cells. This inappropriate recognition causes an immune response that results in the destruction of host tissue. Thus, the immune response, in particular the proliferation of T cells,
Administration of the polynucleotides and polypeptides of the invention that can inhibit differentiation or chemotaxis can be an effective treatment in the prevention of autoimmune disorders.

Examples of autoimmune diseases and disorders that can be treated, prevented and / or diagnosed by the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the present invention include, but are not limited to, one or more of the following: : Autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, autoimmune cytopenias, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, Relapsing polychondritis, rheumatic heart disease, glomerulonephritis (eg, IgA nephropathy), multiple sclerosis, neuritis, uveitis ophthalmitis, multiple endocrine disease, purpura (eg, Henoch-Schönlein) (Henloc
h-Scoenlein) purpura), Reiter's disease, Stiff-Man syndrome,
Autoimmune lung inflammation, autism, Giant-Barre syndrome, insulin-dependent diabetes (
mellitis), and autoimmune inflammatory eyes, autoimmune thyroiditis, hypothyroidism (ie, Hashimoto's thyroiditis), systemic lupus erythematosus, Goodpasture's syndrome, pemphigus, receptor autoimmunity (eg, (a) Graves' disease, (B)
Myasthenia gravis, and (c) insulin resistance), autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, scleroderma with anti-collagen antibodies, mixed connective tissue disease, multiple Myositis / dermatomyositis, pernicious anemia, idiopathic Addison disease, infertility, glomerulonephritis (primary glomerulonephritis and Ig)
A nephropathy), bullous pemphigoid, Sjogren's syndrome, diabetes and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, Other endocrine insufficiency,
Vitiligo, vasculitis, post-myocardial infarction (post-MI), open heart syndrome, hives, atopic dermatitis, asthma, inflammatory myopathy and other inflammatory disorders, granulomatous disorders, degenerative disorders and atrophic Failure.

Which can be treated, prevented and / or diagnosed by the composition of the invention,
Autoimmune disorders (which can occur) include, but are not limited to: rheumatoid arthritis (eg, often characterized by immune complexes in the joints), scleroderma with anti-collagen antibodies (
For example, often characterized by nucleolar antibodies and other nuclear antibodies), mixed connective tissue disease (eg, often characterized by antibodies to soluble nuclear antigens (eg, ribonucleoprotein)), polymyositis (eg, Often characterized by non-histone ANA), pernicious anemia (eg often characterized by antimural cells, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (eg often by humoral and cell-mediated adrenal cytotoxicity) Characterized)
Infertility (eg, often characterized by anti-sperm antibodies), glomerulonephritis (eg, often characterized by glomerular basement membrane antibodies or immune complexes), bullous pemphigoid (eg, in the basement membrane) Often characterized by IgG and complement), Sjogren's syndrome (eg often characterized by multiple tissue antibodies, and / or certain non-histone ANA (SS-B)), diabetes (eg cell-mediated and humoral) Is often characterized by islet cell antibodies), as well as adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (eg, often characterized by β-adrenergic receptor antibodies).

Additional autoimmune disorders that may be treated, prevented, and / or diagnosed using the compositions of the present invention include, but are not limited to: chronic active hepatitis (eg, Often characterized by smooth muscle antibodies), primary biliary cirrhosis (eg often characterized by mitochondrial antibodies), other endocrine insufficiency (eg often in some cases characterized by specific tissue antibodies) ), Vitiligo (eg, often characterized by melanocyte antibodies), vasculitis (eg, often characterized by Ig and complement and / or low serum complement in the vessel wall), post-MI (eg, myocardial antibodies Often characterized by cardiotomy syndrome (eg, often by myocardial antibodies) Characterized), urticaria (eg, often characterized by IgG and IgM antibodies to IgE), atopic dermatitis (eg, often characterized by IgG and IgM antibodies to IgE), asthma (eg, Often characterized by IgG and IgM antibodies against IgE), as well as many other inflammatory, granulotypic, degenerative and atrophic disorders.

In preferred embodiments, autoimmune diseases and disorders and / or conditions associated with the diseases and disorders listed above are used, for example, using an antagonist, or agonist, polypeptide or polynucleotide or antibody of the present invention, Treated, prevented, and / or diagnosed.

In certain preferred embodiments, the polynucleotides, polypeptides, and / or agonists or antagonists of the present invention are used as agents for boosting immunoreactivity in B cell and / or T cell immunocompromised individuals. Can be.

B cell immunodeficiencies that can be restored or treated by administering a polynucleotide or polypeptide of the invention and / or an agonist thereof include, but are not limited to: severe combined immunity Deficiency (SCI
D) -X-linked, SCID-autosome, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infant agammaglobulin blood Disease, acquired agammaglobulinemia, adult-onset agammaglobulinemia, late-onset agammaglobulinemia, aberrant gammaglobulinemia, hypogammaglobulinemia, transient infantile hypogammaglobulinemia, nonspecific Hypogammaglobulinemia, agammaglobulinemia, unclassifiable immunodeficiency (CVI) (acquired), Viscott-Oldrich syndrome (WAS), X-linked immune deficiency with excess IgM, non-with excess IgM X-linked immune deficiency, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), normal Antibody deficiency with Ig or elevated Ig, immune deficiency with thymoma, Ig heavy chain deficiency, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immune deficiency, regression agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic lymphoplasia with immune deficiency-dysgenesis or dysplasia, telangiectasia ataxia Short-limbed dwarfism, X-linked lymphoproliferative syndrome (XLP),
Neserofu syndrome combined immune deficiency with Ig, purine nucleoside phosphorylase deficiency (PNP), MHC class II deletion (deficient lymphocyte syndrome) and severe combined immune deficiency.

T cell dysfunction that can be alleviated or treated by administering a polypeptide or polynucleotide of the present invention and / or an agonist thereof includes, but is not limited to, for example: DiGeorge malformation, thymic growth failure 3rd and 4th pharyngeal sac syndrome, 22q11.2 deficiency, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4 + T lymphocyte depletion, immune deficiency with marked T cell deficiency (unspecified) And unspecified immune deficiencies of cell-mediated immunity. In certain embodiments, a DiGeorge malformation or a condition associated with a DiGeorge malformation is alleviated or treated, for example, by administering a polypeptide or polynucleotide of the invention, or an antagonist or agonist thereof.

Other immunodeficiencies that can be ameliorated or treated by administering the polypeptides or polynucleotides of the present invention, and / or their agonists or antagonists include severe combined immunodeficiency (SCID; eg, X-linked S
CID, autosomal SCID, and adenosine deaminase deficiency), telangiectasia ataxia, Viscott-Aldrich syndrome, short limb (short-
include: dwarfism, X-linked lymphocytosis syndrome (XLP), nezerofu syndrome (eg, purine nucleoside phosphorylase deficiency), MHC class II deficiency. In certain embodiments, telangiectasia ataxia or conditions associated with telangiectasia ataxia can be ameliorated or treated by administering a polypeptide or polynucleotide of the invention, and / or an agonist thereof. .

In certain preferred embodiments, rheumatoid arthritis is treated, prevented, and / or diagnosed using the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the present invention. In another specific embodiment, systemic lupus erythematosus is treated, prevented and / or used with a polynucleotide, polypeptide, antibody and / or agonist or antagonist of the invention.
Or diagnosed. In another particular preferred embodiment, idiopathic thrombocytopenic purpura is treated, prevented and / or diagnosed using the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the present invention.
In another particular preferred embodiment, IgA nephropathy is treated, prevented and / or diagnosed using the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the present invention. In preferred embodiments, autoimmune diseases and autoimmune disorders and / or conditions associated with the diseases and disorders described above are treated, prevented, and / or diagnosed using antibodies to the proteins of the invention.

Similarly, allergic reactions and conditions (eg asthma, especially allergic asthma)
Or other respiratory disorders) can also be treated with the polypeptides, antibodies, polynucleotides, and / or agonists or antagonists thereof,
Can be prevented and / or diagnosed. In addition, these molecules can be used to treat, prevent and / or diagnose anaphylaxis, hypersensitivity to antigenic molecules, or blood group incompatibility.

Furthermore, the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the present invention may also be used to treat, diagnose, and / or prevent inflammatory conditions. Such inflammatory conditions include, but are not limited to, for example: respiratory disorders (eg, asthma and allergies)
Gastrointestinal disorders (such as inflammatory bowel disease); cancer (such as stomach cancer, ovarian cancer, lung cancer,
Bladder cancer, liver cancer, and breast cancer, etc .; CNS disorders (eg, multiple sclerosis, blood-brain barrier permeability, ischemic brain injury, and / or cerebral infarction, traumatic brain injury, neurodegenerative disorders (eg, Parkinson's disease and Alzheimer's disease, etc.), AIDS-related dementia, and prion disease); cardiovascular disorders (eg, atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary bypass complications); and inflammation Many additional diseases, conditions, and disorders (eg, chronic hepatitis (B and C), rheumatoid arthritis, gout, trauma, septic shock, pancreatitis, sarcoidosis, dermatitis, renal ischemia reperfusion injury, Graves' disease, systemic Lupus erythematosus,
Diabetes mellitus (eg, type I diabetes) and allogeneic transplant rejection).

In certain embodiments, the polypeptides, antibodies, polynucleotides of the invention,
And / or their agonists or antagonists may be transplant rejection, graft-versus-host disease, autoimmune and inflammatory diseases (eg, immune complex-induced vasculitis, glomerulonephritis, hemolytic anemia, myasthenia gravis, II It is useful for treating, diagnosing and / or preventing type I collagen-induced arteritis, experimental allergic and hyperacute xenograft rejection, rheumatoid arthritis, and systemic lupus erythematosus (SLE).
Organ rejection is caused by host immune cell destruction of the transplanted tissue by an immune response. Similarly, the immune response is also related to GVHD, where exogenous transplanted immune cells destroy host tissue. Polypeptides, antibodies, or polynucleotides of the invention and / or agonists or antagonists thereof (inhibiting immune responses, particularly T cell activation, proliferation, differentiation, or chemotaxis) prevent organ rejection or GVHD It can be an effective treatment.

Similarly, the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the present invention may also be used to modulate and / or diagnose inflammation. For example, a polypeptide, antibody, or polynucleotide of the present invention, and / or an agonist or antagonist of the present invention may inhibit activation, proliferation and / or differentiation of cells involved in an inflammatory response. Using these molecules, inflammation associated with infection (eg, septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), inflammation associated with ischemia-reperfusion injury, inflammation associated with endotoxin lethality, Inflammation associated with arthritis, inflammation associated with complement-mediated hyperacute rejection, inflammation associated with nephritis, inflammation associated with cytokines or chemokine-induced lung injury, inflammation associated with inflammatory bowel disease, associated with Crohn's disease Treatment, diagnosis, and prognosis of inflammatory conditions in both chronic and acute conditions, including but not limited to inflammation resulting from excessive production of inflammation or cytokines (eg, TNF or IL-1).

The polypeptides, antibodies, polynucleotides and / or agonists or antagonists of the present invention can be used to treat, detect and / or prevent infectious agents. For example, by increasing the immune response, in particular by increasing proliferation, activation and / or differentiation of B cells and / or T cells,
Infectious diseases can be treated, detected, and / or prevented. The immune response can be increased by either increasing an existing immune response or eliciting a new immune response. Alternatively, the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the present invention also directly inhibit infectious agents (as described in the section on application of infectious agents etc.) without the inevitable triggering of an immune response. Can do.

Further preferred embodiments of the present invention include, but are not limited to, the use of polypeptides, antibodies, polynucleotides and / or agonists or antagonists in the following applications:
Boosts the immune system, resulting in increased amounts of one or more antibodies (eg, IgG, IgA, IgM, and IgE) and high affinity antibody production (eg, IgG, Ig
(A, IgM and IgE) and / or animals for increasing immune responses (eg, mice, rats, rabbits, hamsters, guinea pigs, pigs, minipigs, chickens, camels, goats, horses, cows, sheep, Administration to dogs, cats, non-human primates, and humans, most preferably humans).

By means of a reconstituted or partially reconstituted immune system from another animal that cannot produce a functional endogenous antibody molecule or otherwise has an infectious endogenous immune system Administration to animals (including but not limited to the animals listed above, including transgenic animals) capable of producing human immunoglobulin molecules (eg, PCT Publication Number, WO 98/24893, WO 96/34096)
, WO 96/33735, and WO 91/10741).

  A vaccine adjuvant that enhances immune responsiveness to a specific antigen.

  Adjuvant for enhancing tumor specific immune response.

An adjuvant that enhances the antiviral immune response. Antiviral immune responses that can be enhanced using the compositions of the present invention as an adjuvant include viruses and virus-related diseases and conditions described elsewhere herein or otherwise known in the art. In certain embodiments, the composition of the present invention comprises: AID
Used as an adjuvant to enhance an immune response against a virus, disease or condition selected from the group consisting of S, meningitis, dengue, EBV, and hepatitis (eg, hepatitis B). In another specific embodiment, the composition of the invention comprises
The following: HIV / AIDS, RS virus, dengue, rotavirus, Japanese encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus,
Used as an adjuvant to enhance the immune response to a virus, disease or condition selected from the group consisting of rabies, Junin virus, Chikungunya virus, Rift Valley fever, herpes simplex, and yellow fever.

Adjuvant for increasing antibacterial or antifungal immune response. Anti-bacterial or anti-fungal immune responses that can be augmented using the compositions of the present invention as adjuvants include bacteria or fungi, and those described herein or otherwise known in the art Bacteria or fungi associated with the disease or condition. In certain embodiments, the composition of the invention comprises a bacterium or fungus, a disease,
Or used as an adjuvant to increase the immune response to symptoms (selected from the group consisting of tetanus, diphtheria, botulism, and type B meningitis). In certain preferred embodiments, the composition of the present invention comprises a bacterium or fungus, a disease, or a symptom (this is Vibrio cholerae, Myco
bacterium leprae, Salmonella typhi, Sa
Imonella paratyphi, Meisseria meningi
tidis, Streptococcus pneumoniae, Group
B streptococcus, Shigella spp. , Enter
otoxigenic Escherichia coli, Enterohe
morrhagic E.M. coli, Borrelia burgdorfer
i and selected from the group consisting of Plasmodium (malaria)) as an adjuvant to increase the immune response.

Adjuvant to increase antiparasitic immune response. Antiparasitic immune responses that can be augmented using the compositions of the present invention as adjuvants are related to parasites and diseases or conditions described herein or otherwise known in the art. Parasites can be mentioned. In certain embodiments, the compositions of the invention are used as adjuvants to increase the immune response against parasites. In another specific embodiment, the compositions of the invention are used as an adjuvant to increase the immune response against Plasmodium (malaria).

  As a stimulator of B cell responsiveness to pathogens.

  As an activator of T cells.

  As an agent that increases an individual's immune status before receiving immunosuppressive treatment.

  As an agent to induce higher affinity antibodies.

  As a drug to increase serum immunoglobulin levels.

  As a drug to promote recovery of immunocompromised individuals.

  As a drug to boost immune responsiveness among older populations.

As an immune system enhancer before, during, or after bone marrow transplantation and / or other transplantation (eg, allogeneic or exogenous organ transplantation). With respect to transplantation, the compositions of the invention can be administered before, contemporaneously and / or after transplantation. In certain embodiments, the compositions of the invention are administered after transplantation and before initiation of T cell population recovery. In another specific embodiment, the composition of the invention is administered first after transplantation, after the initiation of recovery of the T cell population, but prior to full recovery of the B cell population.

As an agent to boost immune responsiveness among individuals with acquired defects in B cell function. Conditions resulting in an acquired defect in B cell function that can be ameliorated or treated by administering a polypeptide, antibody, polynucleotide and / or agonist or antagonist thereof include HIV infection, AIDS, bone marrow transplantation, and B cells Examples include but are not limited to chronic lymphocytic leukemia (CLL).

As an agent to boost immune responsiveness among individuals with transient immune deficiency. Conditions that result in transient immunodeficiency that can be ameliorated or treated by administering a polypeptide, antibody, polynucleotide and / or agonist or antagonist thereof include viral infections (eg, influenza).
Recovery from recovery, conditions related to malnutrition, recovery from infectious mononucleosis, or conditions related to stress, recovery from measles, recovery from blood transfusion, recovery from surgery, but not limited to Not.

As a regulator of antigen presentation by monocytes, dendritic cells and / or B cells. In one embodiment, the polynucleotide, polypeptide, antibody of the present invention,
And / or an agonist or antagonist enhances or antagonizes antigen presentation in vitro or in vivo. Further, in related embodiments, this enhanced antigen presentation or antagonization may be useful as an anti-tumor treatment or to modulate the immune system.

An individual's immune system is reconstituted with a humoral response (ie, a TH1 cellular response).
2) As a drug for directing the occurrence of

As a means to induce tumor growth and thus make the tumor more sensitive to anti-neoplastic agents. For example, multiple myeloma is a slow cell division (dividing).
) And therefore refractory to virtually all anti-tumor regimens. If these cells are grown more rapidly, their sensitivity profile will likely change.

AIDS, chronic lymphocytic disorders and / or unclassifiable immunodeficiency (Comm
as a stimulator of the production of B cells in pathologies such as on Variable Immunofidelity).

As a treatment for the generation and / or regeneration of lymphoid tissue after surgery, trauma or genetic defects.

As a gene-based therapy for inherited disorders that result in immune deficiencies as observed among SCID patients.

As an antigen for generating antibodies to inhibit or enhance immune-mediated responses to the polypeptides of the invention.

  As a means of activating T cells.

Parasitic diseases affecting monocytes (Leshmania)
As a means of activating monocytes / macrophages to protect against).

As a pretreatment of bone marrow samples before transplantation. Such treatment increases B cell presentation and thus accelerates recovery.

As a means of regulating secreted cytokines induced by the polypeptides of the invention.

Furthermore, the polypeptides or polynucleotides of the present invention and / or agonists thereof can be used to treat or prevent IgE-mediated allergic responses. Such allergic reactions include but are not limited to asthma, rhinitis and eczema.

  All of the above applications can be applied to veterinary medicine.

Antagonists of the present invention include, for example, binding and / or inhibitory antibodies, antisense nucleic acids, and ribozymes. These are expected to negate much of the activity of the above ligands and find clinical or practical applications such as:

As a means to block various aspects of the immune response against foreign factors or self. Examples include autoimmune disorders such as lupus and arthritis, and immune responses against skin allergies, inflammation, bowel disease, injury and pathogens.

As a treatment to prevent B cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and MS.

As an inhibitor of B cell and / or T cell migration in endothelial cells.
This activity disrupts tissue structures or cognate responses and is useful, for example, in disrupting immune responses and blocking sepsis.

  As an inhibitor of graft-versus-host disease or graft rejection.

ALL, Hodgkin's disease, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, plasmacytoma, multiple myeloma, Burkitt's lymphoma and EBV convertibility (EBV-tr
treatment for malignant diseases of B cells and / or T cells such as transformed diseases.

Monoclonal hypergammaglobulinemia of unquantified significance
lgammopathy of undetermined signature
ances) (MGUS), Waldenstrom disease, associated idiopathic monoclonal gammaglobulinemia, and plasmacytoma,
Treatment for chronic hypergammaglobulinemia events.

  Treatment to reduce cell proliferation of large B cell lymphoma.

  Means to reduce B cell and Ig involvement associated with chronic myelogenous leukemia.

  Immunosuppressant.

The polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the present invention can be used to modulate IgE concentrations in vitro or in vivo.

In another embodiment, administration of the polypeptides, antibodies, polynucleotides and / or agonists or antagonists of the invention to treat or prevent IgE-mediated allergic reactions, including but not limited to asthma, rhinitis and eczema. Can be used.

Agonists and antagonists can be used in compositions comprising a pharmaceutically acceptable carrier, eg, as described above.

Agonists or antagonists, for example, in certain autoimmune and chronic inflammatory and infectious diseases, macrophages and their precursors, and neutrophils, basophils, B lymphocytes and some T cell subsets (eg,
Activated T cells and CD8 cytotoxic T cells and natural killer cells) can be used to inhibit polypeptide chemotaxis and activation. Examples of autoimmune diseases are described herein and examples of autoimmune diseases include multiple sclerosis and insulin-dependent diabetes. Antagonists or agonists can also be used to treat infectious diseases, including silicosis, sarcoidosis, idiopathic pulmonary fibrosis, for example, by preventing mononuclear phagocyte recruitment and activation. They can also be used to treat idiopathic eosinophilia syndrome, for example, by preventing eosinophil production and migration. Antagonists or agonists can also be used to treat atherosclerosis, for example, by preventing monocyte infiltration in the arterial wall.

Antibodies against the polypeptides of the invention can be used to treat ARDS.

The agonists and / or antagonists of the present invention also have uses in stimulating wound and tissue repair, stimulating angiogenesis, stimulating repair of vascular or lymphatic diseases or disorders. Furthermore, the agonists and antagonists of the present invention can be used to stimulate mucosal surface regeneration.

In certain embodiments, the polynucleotide or polypeptide, and / or
Alternatively, these agonists are used to treat or prevent disorders characterized by primary or acquired immune deficiency, defective serum immunoglobulin production, recurrent infection and / or immune system dysfunction. In addition, the polynucleotide or polypeptide, and / or agonist thereof may be used in joint, bone, skin and / or parotid gland infection, blood-derived infection (eg, sepsis, meningitis, septic arthritis and / or bone marrow). Flame), autoimmune diseases (eg, autoimmune diseases as disclosed herein), inflammatory disorders, and malignant diseases, and / or any associated with these infections, diseases and / or malignant diseases Disease or disorder or condition (CVID, other primary immunodeficiency, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (eg, severe herpes zoster Herpes) and / or pneumocystis) can be used to treat or prevent.

In another embodiment, the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention are not classified immunodeficiency (C
ommon variable immunity) ("CV
ID ”; also known as“ acquired agammaglobulinemia ”and“ acquired hypogammaglobulinemia ”) or individuals with a subset of this disease are used to treat and / or diagnose.

In certain embodiments, the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the invention treat, diagnose (1) cancer or neoplasm, and (2) autoimmune cells or tissue-related cancer or neoplasm. ,and/
Or it can be used to prevent. In preferred embodiments, the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the present invention conjugated to toxins or radioisotopes as described herein are for acute myeloid leukemia. It can be used for treatment, diagnosis, and / or prevention. In further preferred embodiments, the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention conjugated to toxins or radioactive isotopes, as described herein, are chronic bone marrow. Treating leukemia, multiple myeloma, non-Hodgkin lymphoma, and / or Hodgkin's disease,
Can be used for diagnosis, diagnosis, and / or prevention.

In another specific embodiment, a polynucleotide or polypeptide of the invention and / or an agonist or antagonist is used to select selective Ig.
A deficiency, myeloperoxidase deficiency, C2 deficiency, capillary dilatation ataxia, Di
George malformation, unclassifiable immunodeficiency (CVI), X-linked gamma globulin blood, severe combined immunodeficiency (SCID), chronic granulomatosis (CGD), and Wisk
ot-Aldrich syndrome may be treated, diagnosed, predicted, and / or prevented.

Examples of autoimmune disorders that can be treated or detected include Addison's disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, frequent occurrence Multiple sclerosis, myasthenia gravis, neuritis, ophthalmic pain, bullous pemphigoid, pemphigus, multiple endocrine disease, purpura, Reiter's disease, stiff man syndrome, autoimmune thyroiditis, systemic lupus erythematosus , Autoimmune lung inflammation, Giant-Barre syndrome, insulin-dependent diabetes,
And autoimmune inflammatory eye diseases, including but not limited to.

In preferred embodiments, autoimmune diseases and disorders and / or conditions associated with the diseases and disorders described above are treated, predicted, prevented, and / or diagnosed using antibodies to the polypeptides of the invention.

Used as a factor to boost immune responsiveness among B cell immunodeficient individuals (eg, individuals who have undergone partial or complete splenectomy).

In addition, the polynucleotides, polypeptides and / or antagonists of the present invention can affect apoptosis, and thus the polynucleotides, polypeptides and / or antagonists of the present invention are often associated with increased cell survival or inhibition of apoptosis. Useful for treating various diseases. For example, diseases associated with increased cell survival or inhibition of apoptosis that can be treated or detected by the polynucleotides, polypeptides and / or antagonists of the present invention include: cancer (eg, follicular lymphoma, p53). Cancers with mutations and hormone-dependent tumors (colon cancer, heart tumor, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, gastric cancer, neuroblastoma, mucus Self, including, but not limited to, tumor, myoma, lymphoma, endothelial tumor, osteoblastoma, giant cell tumor, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer)); Immune disorders (eg, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, clones) , Polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (eg, herpesviruses, poxviruses and adenoviruses), inflammation, graft-versus-host disease, acute graft rejection, and Chronic graft rejection. In a preferred embodiment, the polynucleotides, polypeptides, and / or antagonists of the present invention, particularly above listed, the cancer growth, progression, and / or are used to inhibit metastasis of (meta stisis).

Additional diseases or conditions associated with cell survival that can be treated or detected by the polynucleotides, polypeptides and / or antagonists of the present invention include proliferation and / or metastasis of malignant tumors and related disorders such as: Without limitation: leukemia (acute leukemia (eg, acute lymphocytic leukemia, acute myeloid leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia) )), And chronic leukemia (eg, including chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma (eg, Hodgkin's disease and non-Hodgkin's disease), multiple bone marrow Tumors, Waldenstrom macroglobulinemia, heavy chain disease, and solid tumors (sarcomas and carcinomas (eg Fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, hemangiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, periosteum, mesothelioma, Ewing tumor, leiomyosarcoma , Rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer,
Ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, primary bronchial carcinoma, renal cell carcinoma, hepatocyte Cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal cancer, Wilms tumor, cervical cancer, testicular tumor, lung cancer,
Small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ventricular ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroma Gliomas, meningiomas, melanomas, neuroblastomas, and retinoblastomas.

Diseases associated with increased apoptosis that can be treated or detected by the polynucleotides, polypeptides, and / or antagonists of the present invention include: AIDS; neurodegenerative disorders (eg, Alzheimer's disease, Parkinson's disease, muscle Amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or previously related diseases); autoimmune disorders (eg, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease (Behcet) 's d
disease), Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis), myelodysplastic syndromes (eg, aplastic anemia), graft-versus-host disease, ischemic injury (myocardial) Such as those caused by infarction, stroke and reperfusion injury), liver injury (eg, hepatitis related liver injury, ischemia / reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxic-induced liver Diseases (such as those caused by alcohol),
Septic shock, cachexia and anorexia.

Hyperproliferative diseases and / or disorders that can be detected and / or treated by the polynucleotides, polypeptides, and / or antagonists of the present invention include, but are not limited to, neoplasms located in: liver, Abdomen, breast, digestive system, pancreas, peritoneum, endocrine glands (adrenal gland, parathyroid, pituitary gland, testis,
(Ovary, thymus, thyroid), eye, head and neck, nerves (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and urogenital organs.

Similarly, other hyperproliferative disorders can also be treated or detected by the polynucleotides, polypeptides, and / or antagonists of the present invention. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorder, paraproteinemia, purpura, sarcoidosis, Sezary syndrome, Waldenstrom Macroglobulinemia,
Goose disease, histiocytosis, and any other hyperproliferative disease, plus neoplasms found in the organ systems listed above.

(Hyperproliferative disorder)
The polynucleotides or polypeptides, or agonists or antagonists of the present invention may be used to treat, prevent and / or diagnose hyperproliferative diseases, disorders, including neoplasms. The polynucleotides or polypeptides, or agonists or antagonists of the present invention can inhibit the growth of this disorder through direct or indirect interactions. Alternatively, a polynucleotide or polypeptide, or agonist or antagonist of the present invention can proliferate other cells that can inhibit hyperproliferative disorders.

For example, hyperproliferative diseases, disorders and / or conditions may be increased by increasing the immune response, in particular by increasing the antigenic quality of the hyperproliferative disorder, or by proliferating, differentiating or mobilizing T cells. It can be treated, prevented and / or diagnosed. This immune response can be increased by either enhancing an existing immune response or initiating a new immune response. Alternatively, reducing the immune response can also be a method of treating, preventing and / or diagnosing a hyperproliferative disease, disorder and / or condition, such as a chemotherapeutic agent.

Examples of hyperproliferative diseases, disorders and / or conditions that can be treated, prevented and / or diagnosed by a polynucleotide or polypeptide, or agonist or antagonist of the present invention include colon, abdomen, bone, breast, digestive system, Liver, pancreas, peritoneum, endocrine gland (adrenal gland, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nerves (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen , Thorax, and neoplasm located in the genitourinary organs.

Similarly, other hyperproliferative diseases, disorders and / or conditions can also be treated, prevented and / or diagnosed with the polynucleotides or polypeptides, or agonists or antagonists of the present invention. Examples of such hyperproliferative diseases, disorders and / or conditions include hypergammaglobulinemia, lymphoproliferative diseases, disorders and / or conditions, paraproteinemia, purpura, sarcoidosis,
Sezary syndrome, Waldenstrom macroglobulinemia, Goshe disease, histiocytosis, and any other hyperproliferative disease, as well as neoplasms located in the organ system listed above, It is not limited to them.

One preferred embodiment utilizes the polynucleotide of the present invention to
And / or gene therapy with protein fusions or fragments thereof inhibits abnormal cell division.

Accordingly, the present invention provides a method for treating or preventing a cell proliferative disease, disorder and / or condition by inserting a polynucleotide of the present invention into an abnormally proliferating cell. Here, said polynucleotide suppresses said expression.

Another embodiment of the present invention provides a method of treating or preventing a cell proliferative disease, disorder and / or condition in an individual, wherein the method comprises one or more of the present invention on abnormally proliferating cells. Administering an active gene copy of: In a preferred embodiment, the polynucleotide of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding the above polynucleotide. In another preferred embodiment of the invention, a DNA construct encoding a polynucleotide of the invention is inserted into a cell and treated using a retrovirus (or more preferably an adenoviral vector) (this book for reference). G J. Nabel et al., PNAS 1999 96: 324, incorporated herein by reference.
-326). In the most preferred embodiment, the viral vector is defective and does not transform non-proliferating cells, but only proliferating cells. Furthermore, in a preferred embodiment, a polynucleotide of the invention inserted into a proliferating cell alone or with or along with another polynucleotide is then subjected to an external stimulus (ie, Magnetic, specific small molecule, chemical, or drug administration). This stimulus acts on a promoter upstream of the polynucleotide to induce expression of the encoded protein product. In this way, the beneficial therapeutic effects of the present invention can be clearly modulated (ie, to increase, decrease or inhibit the expression of the polynucleotides of the present invention) based on the external stimuli described above.

The polynucleotide of the present invention may be useful in suppressing the expression of oncogenic genes or antigens. “Suppressing the expression of oncogenic genes” refers to suppression of gene transcription, degradation of gene transcripts (pre-massage RNA), inhibition of splicing, disruption of messenger RNA, post-translational modification of proteins. Intended to hinder, destroy proteins, or inhibit normal function of proteins.

For local administration to abnormally proliferating cells, the polynucleotides of the invention can be administered by any method known to those of skill in the art, including transfection, electroporation, cell microinjection, For example, in a vehicle such as a liposome, including, but not limited to, lipofectin, or a naked polynucleotide, or any other method described throughout this specification. The polynucleotides of the present invention can be prepared using known gene delivery systems (eg, retroviral vectors known to those skilled in the art (Gilboa,
J. et al. Virology 44: 845 (1982); Hocke, Nature
320: 275 (1986); Wilson et al., Proc. Natl. Aca
d. Sci. USA. 85: 3014); vaccinia virus system (Chakra)
barty et al., Mol. Cell Biol. 5: 3403 (1985)) or other efficient DNA delivery systems (Yates et al., Nature 313: 812 (
1985))). These references are exemplary only and are incorporated herein by reference. Retroviruses or adenoviruses known to those of skill in the art (eg, the art or in order to deliver specifically to or proliferate cells that are abnormally proliferating and leave non-dividing cells) It is preferred to utilize a delivery system (described herein). Since host DNA replication requires retroviral DNA to integrate, this retrovirus cannot self-replicate due to the lack of required retroviral genes for its life cycle. For the polynucleotides of the present invention, such retroviral delivery systems are utilized to target the genes and constructs described above to abnormally proliferating cells and leave non-dividing normal cells.

The polynucleotides of the invention can be used to produce cell proliferative disorders / internal organs, body cavities, etc. by using an imaging device that is used to guide the needle directly to the disease site.
It can be delivered directly to the disease site. The polynucleotides of the invention can also be administered to the disease site at the time of surgical intervention.

By “cell proliferative disorder” is meant that any human or animal disease or disorder affects any one or any combination of organs, cavities, or body parts. The disease is characterized by a single or multiple local abnormal growth of cells, groups of cells, or tissues, whether benign or malignant.

Any amount of the polynucleotide of the present invention can be administered as long as this amount has a biologically inhibitory effect on the growth of treated cells. Furthermore, more than one polynucleotide of the present invention can be administered at the same site simultaneously. "
“Biologically inhibitory” means partial or total growth inhibition as well as a decrease in the rate of proliferation or growth of cells. Biologically inhibitory doses can be used to assess the effect of a polynucleotide of the invention on the growth of target malignant or abnormally proliferating cells in tissue culture, tumor growth in animals and cell cultures, or It can be determined by any other method known to those skilled in the art.

The present invention further relates to antibody-based therapy, wherein the method is for treating, preventing and / or diagnosing one or more of the described diseases, disorders and / or conditions.
Administering an anti-polypeptide antibody and an anti-polynucleotide antibody to a mammalian (preferably human) patient. Methods for generating anti-polypeptide antibodies and anti-polynucleotide antibodies (polyclonal and monoclonal antibodies) are described in detail elsewhere herein. Such antibodies can be provided in pharmaceutically acceptable compositions as is known in the art or as described herein.

An overview of how the antibodies of the present invention can be used therapeutically includes the polynucleotides or polypeptides of the present invention either locally or systemically in the body, or by direct cytotoxicity (eg, by complement) of the antibody. Binding via mediation (CDC) or effector cell mediated (ADCC)). Some of these approaches are described in more detail below. Given the teaching provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the present invention have, or have developed, a disease, disorder and / or condition of cell proliferation and / or differentiation, as described herein. Is useful for treating, preventing and / or diagnosing. Such treatment includes the step of administering a single dose or multiple doses of the antibody, or a fragment, derivative or conjugate thereof.

The antibodies of the invention may be combined with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (eg, this will act to increase the number or activity of effector cells that interact with the antibody). Can be advantageously used.

The use of high affinity and / or potent in vivo inhibitory and / or neutralizing antibodies against the polypeptides or polynucleotides, fragments or regions thereof of the invention
Preferred for both immunoassays and treatments thereof for diseases, disorders and / or conditions related to (including fragments thereof). Such an antibody, fragment, or region preferably has an affinity for a polynucleotide or polypeptide (including fragments thereof). A preferred binding affinity is 5 ×
10 ⁻⁶ M, 10 ⁻⁶ M, 5 × 10 ⁻⁷ M, 10 ⁻⁷ M, 5 × 10 ⁻⁸ M, 10 ⁻⁸ M, 5 ×
10 ⁻⁹ M, 10 ⁻⁹ M, 5 × 10 ⁻¹⁰ M, 10 ⁻¹⁰ M, 5 × 10 ⁻¹¹ M, 10 ⁻¹¹ M,
5 × 10 ⁻¹² M, 10 ⁻¹² M, 5 × 10 ⁻¹³ M, 10 ⁻¹³ M, 5 × 10 ⁻¹⁴ M, 10 ⁻
Includes affinity with dissociation constants or Kd of less than ¹⁴ M, 5 × 10 ⁻¹⁵ M, and 10 ⁻¹⁵ M.

Furthermore, the polypeptides of the present invention can be either alone, as a fusion protein, or in combination with other polypeptides, either directly or indirectly, as described elsewhere herein. However, it is useful in inhibiting angiogenesis of proliferating cells or tissues. In the most preferred embodiments, the anti-angiogenic effects described above can be achieved indirectly, eg, through inhibition of hematopoietic tumor-specific cells (eg, tumor-associated macrophages) (incorporated herein by reference). J
oseph IB et al., J Natl Cancer Inst, 90 (21):
1648-53 (1998)). Antibodies against the polypeptides or polynucleotides of the present invention may also result in direct or indirect inhibition of angiogenesis (Witte L. et al., Cancer, incorporated herein by reference.
Metastasis Rev. 17 (2): 155-61 (1998)).

The polypeptides of the invention (including protein fusions), or fragments thereof, may be useful in inhibiting proliferating cells or tissues through induction of apoptosis. The polypeptides described above can be, for example, death domain receptor (eg, tumor necrosis factor (TNF) receptor 1, CD95 (Fas), either directly or indirectly, to induce proliferative cell and tissue apoptosis. / APO-
1), TNF receptor-related apoptosis mediator protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptors 1 and 2 (
Schulze-Osthoff K, et al., Incorporated herein by reference.
Eur J Biochem 254 (3): 439-59 (1998))))). Furthermore, in another preferred embodiment of the present invention, the polypeptide described above is expressed through other mechanisms (eg, in the activation of other proteins that activate apoptosis) or the expression of the protein alone or as a small molecule drug. Alternatively, apoptosis can be induced through stimulation either in combination with an adjuvant (eg, apoptonin, galectin, thioredoxin, anti-inflammatory protein) (eg, Mutat Res, incorporated herein by reference). 400 (1-2): 447-55 (1
998), Med Hypotheses. 50 (5): 423-33 (199
8), Chem Biol Interact. Apr 24; 111-112
23-34 (1998)), J Mol Med. 76 (6): 402-12
(1998), Int J Tissue React; 20 (1): 3-15.
(See 1998)).

The polypeptides of the present invention (including protein fusions thereto), or fragments thereof, are useful in inhibiting proliferative cell or tissue metastasis. Inhibition is known as a direct result of administering a polypeptide or an antibody against the polypeptide, or indirectly (eg, to inhibit metastasis, as described elsewhere herein. Which activate the expression of a protein (eg, α4 integrin) (eg, Cur, incorporated herein by reference)
r Top Microbiol Immunol 1998; 231: 125
-41). Such therapeutic effects of the present invention can be achieved either alone or in combination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a composition comprising a polypeptide of the invention (eg, a composition comprising a polypeptide antibody, heterologous nucleic acid, toxin, or prodrug associated with a polypeptide or heterologous polypeptide). Methods are provided for delivery to targeted cells that express the polypeptides of the invention. A polypeptide or polypeptide antibody of the invention can be conjugated to a heterologous polypeptide, heterologous nucleic acid, toxin, or prodrug through hydrophobic, hydrophilic, ionic and / or covalent interactions.

The polypeptides of the invention, protein fusions thereto, or fragments thereof can be directly directed against proliferative antigens and immunogens (eg, when the polypeptide of the invention is “vaccinated” Proliferating, either producing an immune response to respond to the immunogen) or indirectly (eg, in activating expression of a protein (eg, chemokine) known to enhance the immune response) It is useful in enhancing the immunogenicity and / or antigenicity of living cells or tissues.

(Cardiovascular disorder)
The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used to treat, prevent and / or diagnose cardiovascular diseases, disorders and / or conditions, including peripheral arterial diseases such as limb ischemia. .

Cardiovascular diseases, disorders and / or conditions include arterial fistula (arteri)
o-arterial fistula), arteriovenous fistula, cerebral arteriovenous congenital anomalies, congenital heart defects, pulmonary atresia, and scimitar syndrome. Congenital heart defects include aortic constriction, trilobular heart, coronary vascular malformations
el anomalies), crossed heart, right thoracic heart, patent ductus arterial (patent)
Ductus arteriosus), Ebstein malformation, Eisenmenger complex, left ventricular dysgenesis syndrome, left thoracic heart, tetralogy of Fallot, macrovascular transposition, biventricular right ventricular origin, tricuspid atresia, arterial canal Rest and heart septal defect
septal defects (eg, aortopulmonary septal defect)
topulomary septal defect, endocardial floor deficiency, rytanbache syndrome, trilogy of Fallot, ventricular septal defect (ventricu)
lar heart septal defects)).

Cardiovascular diseases, disorders and / or conditions may also include arrhythmia, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis ( (Including bacterial), cardiac aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture (Post-instruction heart structure),
Ventricular septal rupture, heart valve disease, myocardial disease, myocardial ischemia, endocardial fluid effusion, epicarditis (including infarct and tuberculosis), pneumocardiosis, postpericardiotomy syndrome, right heart disease, Rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications (cardiovascu)
lar pregnancy complications, scimitar syndrome, cardiovascular syphilis, and cardiovascular tuberculosis
heart disease such as culosis).

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes syndrome, leg block, sinoatrial block, long QT syndrome (long
QT syndrome, accessory contraction, Lone-Gannong-Levine syndrome, Mahheim-type pre-excita
(tion syndrome), Wolf-Parkinson-White syndrome, sinus failure syndrome, tachycardia, and ventricular fibrillation. Tachycardia includes paroxysmal tachycardia,
Supraventricular tachycardia, ventricular proper rhythm promotion, atrioventricular nodule reentry tachycardia
cellular nodal reentry tachycardia), ectopic atrial tachycardia, ectopic junction tachycardia, sinoatrial nodal reentry tachycardia
dal reentry tachycardia), sinus tachycardia, torsade
De pointe, and ventricular tachycardia.

Heart valve diseases include aortic valve dysfunction, aortic stenosis, heart noise (hea)
r murmurs), aortic valve prolapse, mitral prolapse, tricuspid prolapse, mitral dysfunction, mitral stenosis, pulmonary atresia, pulmonary valve dysfunction, pulmonary stenosis, tricuspid Include atresia, tricuspid dysfunction, and tricuspid stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, subvalvular aortic stenosis, subvalvular pulmonary stenosis, restraint cardiomyopathy, Chagas cardiomyopathy, endocardial fiber elasticity Disease, endocardial myocardial fibrosis, Keynes syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemia includes angina, coronary aneurysm, coronary atherosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction, and myocardial stunnin.
and coronary artery disease such as g).

Cardiovascular diseases also include aneurysms, angiogenesis abnormalities, hemangiomatosis, bacterial hemangioma symptoms, Hippel-Lindau disease (Hippel-Lindau Disease)
), Klipel-Tornonnay-Weber syndrome, Sturge-Weber syndrome,
Vasomotor edema, aortic disease, Takayasu arteritis, aortitis, Lleish syndrome,
Arterial occlusion disease, arteritis, arteritis, nodular polyarteritis, cerebrovascular disease, disorder and / or condition, diabetic vasculopathy, diabetic retinopathy, embolism, thrombosis, tip Erythema, hemorrhoids, hepatic vein occlusion disorder, hypertension, hypotension, ischemia, peripheral vascular disorder, phlebitis, pulmonary vein occlusion disease, hypertension, hypotension, ischemia, peripheral vascular disease, phlebitis, pulmonary vein occlusion disease , Lehno's disease, CREST syndrome, retinal vein occlusion, scimiter syndrome, superior vena cava syndrome, telangiectasia, telangiectasia ataxia, hereditary hemorrhagic telangiectasia, varicocele of the vagina, dilated serpentine Examples include vascular diseases such as veins, varicose ulcers, vasculitis, and venous dysfunction.

Aneurysms include dissecting aneurysms, pseudoaneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, cardiac aneurysms, and iliac aneurysms .

Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasia, mesenteric vascular occlusion, moyamoya disease, renal artery occlusion, retinal artery occlusion, and obstructive thromboangiitis Is mentioned.

Cerebrovascular diseases, disorders and / or conditions include carotid artery disease, cerebral amyloid vascular disorder, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous congenital anomaly, cerebral artery disease, cerebral embolism And thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage (subara
xhnoid hemorrhage), cerebral infarction, cerebral ischemia (including transient)
, Subclavian artery steal syndrome, periventricular softening
eukomalacia), vascular headache, cluster headache, migraine, and vertebrobasilar dysfunction.

Embolisms include air embolism, amniotic fluid embolism, cholesterol embolism, toe cyanosis syndrome, fat embolism, pulmonary embolism, and thromboembolism. Thrombosis includes coronary artery thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis,
Sinus thrombosis, Wallenberg syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, partition syndrome (compartme
nt syndrome, anti-compartment syndrome
tment syndrome), myocardial ischemia, reperfusion injury, and peripheral limb ischemia. Vasculitis includes aortitis, arteritis, and Behce.
t) Syndrome, Churg-Strauss Syndrome, Mucocutaneous Lymph Node Syndrome, Obstructive Thrombotic Vasculitis, Hypersensitivity Vasculitis, Schönlein-Henoho Purpura (Schoenl)
ein-Henoch purpura), allergic dermatovasculitis and Wegener's granulomatosis.

The polynucleotides or polypeptides, or agonists or antagonists of the present invention are particularly effective for the treatment of dangerous limb ischemia and coronary artery disease.

Polypeptides can be administered using any method known in the art, including direct needle injection at the delivery site, intravenous injection, topical administration,
Catheter injection, biolistic injection, particle accelerator, gelfoam sponge depot, other commercially available depot materials, osmotic pumps, oral or suppository solid pharmaceutical formulations, intracanal decanting or topical application, aerosol delivery However, it is not limited to these. Such methods are known in the art.
The polypeptides of the present invention are therapeutic agents (Theraps, described in more detail below).
eutic). Methods for delivering the polynucleotides of the invention are described in more detail herein.

(Anti-angiogenic activity)
The naturally occurring equilibrium between the stimulatory factor and the inhibitor of angiogenesis is the equilibrium that dominates the inhibitory effect. Rastinejad et al., Cell 56
: 345-355 (1989). In rare cases where neovascularization occurs under normal physiological conditions (eg, wound healing, organ regeneration, embryonic development, and female reproductive processes), angiogenesis is tightly regulated and spatial and temporal Determined. Under the conditions of pathological angiogenesis (eg, characterizing solid tumor growth), these modulations cannot be controlled. Unregulated angiogenesis becomes pathological and maintains the progression of many neoplastic and non-neoplastic diseases. Many serious diseases are dominated by abnormal neovascularization, including solid tumor growth and metastasis, arthritis, some types of eye disorders and psoriasis. For example, Mo
ses et al., Biotech. 9: 630-634 (1991); Folkman
Et al. Engl. J. et al. Med. 333: 1757-1763 (1995);
Auerbach et al. Microvasc. Res. 29: 401-411
(1985); Folkman, Advances in Cancer Re
search, Klein and Weinhouse, Academic P
res, New York, 175-203 (1985); Patz, Am
. J. et al. Optalmol. 94: 715-743 (1982); and Fol.
See review by kman et al., Science 221: 719-725 (1983). In many pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data has been accumulated to suggest that solid tumor growth is dependent on angiogenesis. Folkman and Klagsbrun, S
science 235: 442-447 (1987).

The present invention provides for the treatment of diseases, disorders and / or conditions associated with neovascularization by administration of the polynucleotides, polypeptides, agonists and / or antagonists of the present invention. Malignant and metastatic conditions that can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the present invention include the malignant tumors, solid tumors, and cancers described herein, as well as others known in the art. (For a review of such disorders, see Fishm
an et al., Medicine, 2nd edition, J. Am. B. Lippincott Co. ,
Philadelphia (1985)), but is not limited thereto. Accordingly, the present invention treats angiogenesis related diseases and / or disorders comprising administering to a subject in need a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and / or agonist of the present invention. Providing methods for prevention and / or diagnosis. For example, polynucleotides, polypeptides, antagonists and / or agonists can be utilized in a variety of additional ways to therapeutically prevent cancer or tumors. Cancers that can be treated, prevented and / or diagnosed with polynucleotides, polypeptides, antagonists and / or agonists include prostate cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, pancreatic cancer, laryngeal cancer, esophageal cancer, testicular cancer, liver Solid tumors including cancer, parotid cancer, bile duct cancer, colon cancer, rectal cancer, cervical cancer, uterine cancer, endometrial cancer, kidney cancer, bladder cancer, thyroid cancer; primary tumor and metastasis; melanoma; Glioblastoma; capage sarcoma; leiomyosarcoma; non-small cell lung cancer;
Colorectal cancer; advanced malignant disease; and tumors arising from blood (eg, leukemia), but are not limited to these. For example, polynucleotides, polypeptides, antagonists and / or agonists can be delivered locally to treat cancers such as skin cancer, head and neck tumors, breast tumors and Kaposi's sarcoma.

In yet other aspects, polynucleotides, polypeptides, antagonists and / or agonists can be utilized to treat, prevent and / or diagnose bladder cancer surface forms, for example, by intravesical administration. The polynucleotide, polypeptide, antagonist and / or agonist can be delivered directly to the tumor or near the tumor site via injection or catheter. Of course, as the skilled artisan will appreciate, the appropriate mode of administration will vary with the cancer to be treated. Other modes of delivery are discussed herein.

Polynucleotides, polypeptides, antagonists and / or agonists may be useful in treating, preventing and / or diagnosing other diseases, disorders and / or conditions (including angiogenesis) in addition to cancer. . These disorders, diseases and / or conditions include, but are not limited to:
Benign tumors (eg, hemangiomas, acoustic neuromas, neurofibromas, trachoma, and purulent granulomas); atherosclerotic plaques; ocular angiogenic diseases (eg, diabetic retinopathy, immature retinopathy, macular degeneration, cornea) Transplant rejection, neovascular glaucoma, post-lens fiber growth, rubeosis, retinoblastoma, uveitis and pterygium of the eye (Pter
ygia) (abnormal vascular proliferation)); rheumatoid arthritis; psoriasis; delayed wound healing;
Endometriosis; Angiogenesis; Granulation; Hyperplastic scar (keloid); Fracture fracture; Scleroderma; Trachoma; Vascular adhesion; Myocardial neovascularization; Coronary collateral
collaterals); cerebral collateral branches; arteriovenous malformation; ischemic limb angiogenesis; Osler-Webber syndrome; plaque neovascularization; telangiectasia; hemophilic joint; Fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.

For example, in one aspect of the invention, treating, preventing and preventing hyperplastic scars and keloids comprising administering to the hyperplastic scar or keloid a polynucleotide, polypeptide, antagonist and / or agonist of the invention. /
Alternatively, a method for diagnosing is provided.

In one embodiment of the invention, polynucleotides, polypeptides, antagonists and / or agonists are injected directly into hyperplastic scars or keloids to prevent the progression of these lesions. This treatment is particularly valuable in preventive treatment of conditions known to result in the development of hyperplastic scars and keloids (eg burns), and preferably the time during which the proliferative phase proceeds (of the initial injury) After about 14 days) but before the development of hyperplastic scars or keloids. As noted above, the present invention also treats, prevents and / or treats ocular neovascular diseases, including corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, post-lens fiber growth and macular degeneration. Or provide a method for diagnosis.

Furthermore, the polynucleotides and polypeptides of the invention (agonists and / or
Ocular diseases, disorders and / or conditions associated with neovascularization that can be treated with (including antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblast Associated with cytomas, post-lens fibroproliferation, uveitis, retinopathy of prematurity, macular degeneration, corneal transplant neovascularization, and other ocular inflammatory diseases, ocular tumors, and choroidal or iris neovascularization Disease. For example, Waltman et al., Am. J. et al. Ophthal. 85: 704
710 (1978) and Gartner et al. Surv. Ophthal. 2
2: 291- 312 (1978).

Accordingly, in one aspect of the invention, corneal neovascularization (corneal transplantation neoplasia) comprising administering to a patient a therapeutically effective amount of a compound (described above) to the cornea such that blood vessel formation is inhibited. Methods are provided for treating ocular neovascular diseases, including angiogenesis). Briefly, the cornea is a tissue that normally lacks blood vessels. However, in certain pathological conditions, capillaries can extend from the pericorneal vascular plexus to the cornea. When the cornea is vascularized, the cornea is also clouded, resulting in a loss of patient vision. If the cornea becomes completely opaque, it can be completely loss of vision. A wide variety of diseases, disorders and / or conditions can result in, for example, corneal neovascularization, including: corneal infections (eg, trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (Eg, graft rejection and Stevens-Johnson syndrome), alkaline burns, trauma, inflammation (due to any cause), toxicity and nutrient deficiencies, and complications of wearing contact lenses.

In a particularly preferred embodiment of the present invention, it can be prepared for topical administration in saline (in combination with any preservatives and antibacterial agents commonly used in ophthalmic preparations) and administered in eye drop form Can be done. Solutions or suspensions are prepared in pure form and can be administered several times a day. Alternatively, an anti-angiogenic composition prepared as described above can also be administered directly to the cornea. In a preferred embodiment, the anti-angiogenic composition is prepared with a mucoadhesive polymer that binds to the cornea. In a further embodiment, the anti-angiogenic factor or anti-angiogenic composition is
It can be used as an adjunct to conventional steroid therapy. Local treatment may also be useful prophylactically in corneal lesions known to have a high potential to induce angiogenic responses (eg, chemical burns). In these cases, treatment (possibly combined with steroids) can be started immediately to help prevent subsequent complications.

In other embodiments, the compounds described above can be injected into the corneal stroma directly by an ophthalmologist under microscopic guidance. The preferred injection site may vary in the form of individual lesions, but the goal of administration is to place the composition on the advancing surface of the vasculature (ie, distributed between the blood vessels and the normal cornea). Is). In most cases, this is the perimeter (pe) to “protect” the cornea from the advancing blood vessels.
(ririmbic) including corneal injection. This method can also be utilized immediately after corneal injury to prevent prophylactic corneal neovascularization. In this situation, the substance can be dispersed and injected into the perilimbic cornea between the corneal lesion and its unwanted potential blood supply edge. Such methods can also be utilized in a similar manner to prevent capillary invasion of the transplanted cornea. In sustained release form, infusion may only be required 2-3 times a year. Steroids can also be added to the infusion solution to reduce inflammation resulting from the injection itself.

In another aspect of the invention, treating neovascular glaucoma comprising administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and / or agonist so as to inhibit blood vessel formation. A method for doing so is provided. In one embodiment, the compound can be administered topically to the eye to treat an early form of neovascular glaucoma. In other embodiments, the compound can be implanted by injection into the area of the anterior chamber angle. In other embodiments, the compound can also be placed at any location so that the compound is continuously released into the aqueous humor. In another aspect of the invention,
A method for treating proliferative diabetic retinopathy comprising administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and / or agonist such that angiogenesis is inhibited in a patient Is provided.

In a particularly preferred embodiment of the invention, proliferative diabetic retinopathy is treated by infusion into the aqueous humor or vitreous to increase the local concentration of polynucleotides, polypeptides, antagonists and / or agonists in the retina. Can be done. Preferably, this treatment should be initiated before the acquisition of a serious disease that requires photocoagulation.

In another aspect of the present invention, post-lens fiber growth comprising administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and / or agonist such that blood vessel formation is inhibited in a patient. A method for treating a disease is provided. The compound can be administered locally via intravitreal injection and / or via intraocular implantation.

In addition, disorders that can be treated, prevented, and / or diseased with this polynucleotide, polypeptide, agonist and / or agonist include, but are not limited to: hemangiomas, arthritis, psoriasis, hemangiofibromas, Atherosclerotic plaque, delayed wound healing, granulation, hemophilic joint, hyperplastic scar,
False joint fractures, Osler-Weber syndrome, pyogenic granulomas, scleroderma, trachoma, and vascular adhesion.

In addition, disorders and / or conditions that can be treated with this polynucleotide, polypeptide, agonist and / or agonist include, but are not limited to: solid tumors, blood born tumors (eg, Leukemia), tumor metastasis, capage sarcoma, benign tumors (eg, hemangiomas, acoustic neuromas, neurofibromas, trachoma, and pyogenic granulomas), rheumatoid arthritis,
Psoriasis, ocular angiogenic diseases (eg diabetic retinopathy, retinopathy of prematurity, macular degeneration,
Corneal transplant rejection, neovascular glaucoma, post-lens fibroproliferation, rubeosis, retinoblastoma, and uveitis), delayed wound healing, endometriosis,
Angiogenesis, granulation, hyperplastic scar (keloid), pseudoarticular fracture, scleroderma, trachoma, vascular adhesion, myocardial neovascularization, coronary colla
tals), cerebral collateral branch, arteriovenous malformation, ischemic limb angiogenesis, Osler-Weber syndrome, plaque neovascularization, telangiectasia, hemophilic joint,
Hemangiofibroma, fibromyogenic dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control drugs (by controlling menstruation, by preventing angiogenesis necessary for embryo implantation), pathogenesis Sexual results (eg, Roche mi
naria quintosa), ulcer (Helicobacter pyro)
ri), diseases with angiogenesis such as bartonellosis and bacterial hemangioma symptoms).

In one aspect of the birth control method, an amount of compound sufficient to block embryo implantation is administered before or after sexual intercourse and fertilization occurs, and thus an effective method of birth control, perhaps “post-hoc”. (Morning after) "method is provided. Polynucleotides, polypeptides, agonists and / or agonists can also be used in controlling menstruation or administered either as peritoneal lavage fluid in the treatment of endometriosis or for peritoneal transplantation.

The polynucleotides, polypeptides, agonists and / or agonists of the present invention can be incorporated into surgical sutures to prevent stitch granulomas.

A polynucleotide, polypeptide, agonist and / or agonist is
It can be utilized in a wide variety of surgical procedures. For example, in one aspect of the invention, the composition (eg, in the form of a spray or film) separates normal surrounding tissue from malignant tissue and / or prevents the spread of disease to the surrounding tissue. To do so, it can be utilized to coat or spray the area prior to removal of the tumor. In other aspects of the invention, the composition (eg, in the form of a spray) is delivered via an endoscopic procedure to coat a tumor or to inhibit angiogenesis at a desired location. obtain. In still other aspects of the present invention, a surgical mesh coated with the anti-angiogenic composition of the present invention can be utilized in any procedure in which a surgical mesh can be utilized. For example, in one embodiment of the invention, a surgical mesh raden (lad) having an anti-angiogenic composition.
en) can be utilized during abdominal cancer resection surgery (eg, after colectomy) to provide support for the structure and to release a certain amount of anti-angiogenic factors.

In a further aspect of the invention, a method for treating a tumor resection site is provided. The method comprises administering a polynucleotide, polypeptide, antagonist and / or agonist to the resected margin of the tumor after resection to inhibit local recurrence of cancer and neovascularization at that site. Is included. 1 of the present invention
In one embodiment, the anti-angiogenic compound is administered directly to the site of tumor resection (e.g., using the anti-angiogenic compound, swabbed, brushed, or Applied by other methods of coating).
Alternatively, the anti-angiogenic compound can be incorporated into a known surgical paste prior to administration. In a particularly preferred embodiment of the invention, the anti-angiogenic compound is applied after hepatectomy for malignant disease and after neurosurgical surgery.

In one aspect of the present invention, the polynucleotides, polypeptides, agonists and / or antagonists of the present invention are at the margin of resection of a wide variety of tumors, including breast tumors, colon tumors, brain tumors, and liver tumors. Can be administered. For example, in one embodiment of the invention, an anti-angiogenic compound can be administered at the site of a neurological tumor after resection, so that the formation of new blood vessels at that site is inhibited.

The polynucleotides, polypeptides, agonists and / or antagonists of the invention can also be administered with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: anti-invasive factors,
Retinoic acid and its derivatives, paclitaxel, suramin, tissue inhibitor of metalloproteinase-1, tissue inhibitor of metalloproteinase 2, plasminogen activator inhibitor 1, plasminogen activator inhibitor 2
, And various forms of light “group d” transition metals.

Lighter “group d” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species can form transition metal complexes. Suitable complexes of the above transition metal species include oxo transition metal complexes.

Representative examples of vanadium complexes include oxo vanadium complexes (eg, vanadate and vanadyl complexes). Suitable vanadate complexes include metavanadate and orthovanadate complexes (eg, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate). Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate (including vanadyl sulfate hydrate (eg, vanadyl sulfate monohydrate and vanadyl sulfate trihydrate)).

Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate complexes and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxomolybdenum complexes include molybdate complexes, molybdenum oxide complexes, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrate, sodium molybdate and its hydrate, and potassium molybdate and its hydrate. Suitable molybdenum oxides include molybdenum oxide (VI), molybdenum oxide (
VI) and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include, for example, hydroxo derivatives derived from glycerol, tartaric acid and sugars.

A wide variety of other anti-angiogenic factors may also be utilized in the context of the present invention. Representative examples include: platelet factor 4; protamine sulfate; sulfated chitin derivatives (prepared from queen crab shells) (Mur
ata et al., Cancer Res. 51: 22-26, 1991); sulfated polysaccharide peptidoglycan complex (SP-PG) (the function of this compound can be enhanced by the presence of steroids such as estrogen and tamoxifen citrate); staurosporine; Regulators of substrate metabolism (eg proline analogues,
Cishydroxyproline, d, L-3,4-dehydroproline, thiaproline,
α, α-dipyridyl, including aminopropionitrile fumarate); 4-propyl-5- (4-pyridinyl) -2 (3H) -oxazolone; methotrexate;
Hetoline; interferon; 2 macroglobulin-serum; C
hIMP-3 (Pavloff et al., J. Bio. Chem. 267: 17321
-17326, 1992); chymostatin (Tomkinson et al., Bioch).
em J.M. 286: 475-480, 1992); cyclodextrin tetradecasulfate; eponemycin; camptothecin; fumagillin (Ingber
Et al., Nature 348: 555-557, 1990); gold sodium thiomalate ("GST"; Matsubara and Ziff, J. Clin. In
vest. 79: 1440-1446, 1987); anti-collagenase-serum; α2-antiplasmin (Holmes et al., J. Biol. Chem. 262 (4).
): 1659-1664, 1987); Bissantren (National Ca)
ncer Institute); lobenzalite disodium (N- (2)-
Carboxyphenyl-4-chloroanthronyl acid
ilic acid) disodium, or “CCA”; Takeuchi et al.,
Agents Actions 36: 312-316, 1992); thalidomide; Angostotic steroids; AGM-1470; carboxyaminoimidazolide; and metalloproteinase inhibitors (eg, BB94).

(Disease at the cellular level)
Diseases associated with increased cell survival or inhibition of apoptosis that can be treated, prevented and / or diagnosed by the polynucleotides or polypeptides, and / or antagonists or agonists of the present invention include: cancer (eg, , Follicular lymphoma, carcinoma with p53 mutations, and hormone-dependent tumors, including but not limited to: colon cancer, heart tumor, pancreatic cancer,
Melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, gastric cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelial tumor, osteoblastoma, osteoclastoma, Osteosarcoma,
Chondrosarcoma, adenoma, breast cancer, prostate cancer, capage sarcoma and ovarian cancer); autoimmune diseases, disorders and / or conditions (eg, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease Polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis), and viral infections (eg, herpesviruses, poxviruses and adenoviruses), inflammation, graft-versus-host disease, acute graft rejection, As well as chronic graft rejection. In preferred embodiments, the polynucleotides or polypeptides and / or agonists or antagonists of the present invention are used to inhibit cancer growth, progression, and / or metastasis, particularly in those listed above. The

Additional diseases or conditions associated with increased cell survival that can be treated, prevented, and / or diagnosed with the polynucleotides or polypeptides, or agonists or antagonists of the present invention include malignant disease progression and / or metastasis and: Related disorders include, but are not limited to: leukemia (acute leukemia (eg, acute lymphocytic leukemia, acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and Including erythroleukemia) and chronic leukemia (eg, chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (eg, Hodgkin's disease and non-Hodgkin's disease), multiple Myeloma, Waldenstrom macroglobulinemia, heavy chain disease, and solid tumors ( Anatta, but are not limited to: sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
Endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial tumor, periosteum, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
Pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary carcinoma, cystadenocarcinoma, medullary carcinoma, primary bronchial carcinoma, kidney Cell carcinoma,
Hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic cancer, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, Medulloblastoma, craniopharyngioma, ventricular ependymoma, pineal gland, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and Retinoblastoma))).

Diseases associated with increased apoptosis that can be treated, prevented, and / or diagnosed with the polynucleotides or polypeptides, and / or agonists or antagonists of the present invention include: AIDS; neurodegenerative diseases, disorders, and / or Or condition (eg, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or related disease); autoimmune disease, disorder and / or condition (eg, multiple disease) Sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis), spinal dysplasia syndrome (eg, aplastic anemia) ), Graft versus host disease, ischemic injury (myocardial infarction) , Like those caused by stroke and reperfusion injury),
Liver injury (eg, hepatitis-related liver injury, ischemia / reperfusion injury, cholestasis (cho
lestosis (bile duct injury) and liver cancer); toxic-induced liver disease (such as caused by alcohol), septic shock, cachexia and anorexia.

(Wound healing and epithelial cell proliferation)
In accordance with yet a further aspect of the present invention, the polynucleotides or polypeptides, and / or agonists or antagonists of the present invention for therapeutic purposes,
For example, a process is provided for utilization to stimulate epithelial cell proliferation and basal keratinocytes for wound healing purposes and to stimulate hair follicle production and skin wound healing. A polynucleotide or polypeptide of the invention,
And agonists or antagonists may be clinically useful in stimulating wound healing, including: surgical wounds, resection wounds, deep wounds (including dermal and epidermal damage), ocular tissue wounds, Dental tissue wounds, oral wounds, diabetic ulcers, skin ulcers, elbow ulcers, arterial ulcers, venous stasis ulcers, heat exposure or chemical burns, and other abnormal wound healing Complications associated with a condition (eg, uremia, malnutrition, vitamin deficiency, and / or systemic treatment with steroids, radiotherapy and antitumor drugs and antimetabolites. Polynucleotides or polypeptides of the invention, As well as agonists or antagonists can be used to promote skin recovery after skin loss.

The polynucleotides or polypeptides and / or agonists or antagonists of the present invention can be used to increase the attachment of skin grafts to the wound bed and to stimulate re-epithelialization from the wound bed. . The following are types of grafts in which the polynucleotides or polypeptides, agonists or antagonists of the present invention can be used to increase adhesion to a wound bed:
Autograft, artificial skin, allograft (autograft), autograft, autoepidermic graft, avascular
lar) grafts, Blair-Brown grafts, bone grafts, embryonic tissue grafts, dermal grafts, delayed grafts, skin grafts, epidermal grafts, fascia grafts, full thickness skin grafts, xenografts (Heterologous graph), xenograft (xenog)
raft), allograft, proliferative graft, lamellar graft, reticular graft, mucosal graft, orier-teirsch graft, omental graft, patch transplantation Pieces, stem-like grafts, full-thickness grafts, split-skin grafts
kin graft), thick split graft.
The polynucleotides or polypeptides and / or agonists or antagonists of the present invention can be used to promote skin strength and to improve the appearance of aged skin.

The polynucleotides or polypeptides and / or agonists or antagonists of the present invention can also be used for hepatocyte proliferation and lung, breast, pancreas, stomach, small intestine (
small intesting) and changes in epithelial cell proliferation in the large intestine. A polynucleotide or polypeptide of the invention,
And / or agonists or antagonists may be epithelial cells (eg, sebocytes, hair follicles, liver parenchymal cells, alveolar epithelial cells type II).
II pneumocyte), mucin producing goblet cells, and other epithelial cells, and their ancestors contained within the skin, lung, liver, and gastrointestinal tract). The polynucleotide or polypeptide of the invention, agonist and / or
Alternatively, the antagonist can promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

The polynucleotides or polypeptides and / or agonists or antagonists of the present invention can also be used to reduce intestinal toxic side effects resulting from irradiation, chemotherapy treatment or viral infection. The polynucleotides or polypeptides and / or agonists or antagonists of the present invention may have a cytoprotective effect on the small intestinal mucosa. The polynucleotides or polypeptides and / or agonists or antagonists of the present invention may also stimulate the healing of mucositis (oral mucosa) resulting from chemotherapy and viral infection.

The polynucleotides or polypeptides and / or agonists or antagonists of the present invention provide sufficient regeneration of the skin (ie, hair follicles, sweat glands, and sebaceous gland regrowth) in full and partial thickness skin defects, including burns. ), Which can be further used in the treatment of other skin defects such as psoriasis. The polynucleotides or polypeptides, and agonists and / or antagonists of the present invention provide for epidermolysis bullosa, an underlying dermis that produces frequent open and painful blisters by promoting re-epithelialization of these lesions. It can be used to treat defects in epidermal adhesion. The polynucleotides or polypeptides, and agonists or antagonists of the present invention also treat gastric and duodenal ulcers and aid healing by the scar formation of the mucosal lining and the more rapid regeneration of the mucosa of the gland and duodenal mucosa Can be used for. Inflammatory bowel disease (
For example, Coulomb disease and ulcerative colitis) are diseases that cause disruption of the mucosal surface of the small or large intestine, respectively. Therefore, polynucleotides or polypeptides, and / or agonists or antagonists of the invention, prophylactic resurfacing of the mucosal surface (resu r facing) to promote, to aid more rapid healing, and the progression of inflammatory bowel disease Can be used to Treatment with the polynucleotides or polypeptides, agonists and / or antagonists of the present invention is expected to have a significant effect on the production of mucosa throughout the gastrointestinal tract, and the intestinal mucosa has been ingested or operated on. Can be used to protect against later harmful substances. The polynucleotides or polypeptides and / or agonists or antagonists of the present invention can be used to treat diseases associated with expression of the polynucleotides of the present invention.

Furthermore, the polynucleotides or polypeptides and / or agonists or antagonists of the present invention can be used to prevent and cure lung damage resulting from various pathological conditions. Growth factors such as polynucleotides or polypeptides of the invention and / or agonists or antagonists can stimulate proliferation and differentiation and prevent alveolar and bronchial (to prevent or treat acute or chronic lung injury) brochiola) may promote epithelial repair. For example, resulting in necrosis of the bronchial epithelium and alveoli (a l veoli), emphysema (which results in a progressive loss of alveoli) and inhalation injuries (i.e., resulting from inhalation and burns smoke) are present The polynucleotides or polypeptides and / or agonists or antagonists of the invention can be effectively treated. Also, the polynucleotides or polypeptides and / or agonists or antagonists of the present invention can be used to stimulate the proliferation and differentiation of alveolar epithelial cell type II, which can be used for vitreous membrane disease in premature infants (eg, May help treat or prevent diseases such as infant respiratory distress syndrome and bronchopulmonary dysplasia.

The polynucleotides or polypeptides and / or agonists or antagonists of the present invention can stimulate the proliferation and differentiation of hepatocytes and therefore liver diseases and conditions (eg, fulminant liver failure caused by cirrhosis, hepatitis virus and toxicity Substances (ie acetaminophen, carbon tetrachloride (carbon
can be used to alleviate or treat liver injury) caused by tetrahololide), and other liver toxins known in the art.

Furthermore, the polynucleotides or polypeptides and / or agonists or antagonists of the present invention can be used to treat or prevent the development of diabetes mellitus. In patients newly diagnosed with type I and type II diabetes, if some islet cell function remains, the polynucleotide or polypeptide and / or agonist or antagonist of the present invention may be It can be used to maintain its islet function so as to mitigate, delay or prevent expression. The polynucleotides or polypeptides of the present invention, as well as agonists or antagonists, can also be used as an aid in islet cell transplantation to improve or promote islet cell function.

(Neurological disease)
Nervous system diseases, disorders and / or conditions that can be treated, prevented and / or diagnosed using the compositions of the invention (eg, polypeptides, polynucleotides and / or agonists or antagonists) include axonal cutting, Nervous system injuries and diseases that cause either neuronal loss or degeneration, or demyelination,
Examples include but are not limited to faults and / or conditions. Nervous system lesions that can be treated, prevented and / or diagnosed in patients (including human patients and non-human mammalian patients) according to the present invention include the following central nervous systems (including spinal cord, brain):
Or any lesion of the peripheral nervous system, including but not limited to: (
1) ischemic lesions (where oxygen deficiency in part of the nervous system causes neuronal damage or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia); (2) Traumatic lesions (including lesions caused by physical injury or associated with surgery, such as lesions that cut off parts of the nervous system, or compression lesions)
(3) malignant lesions (wherein part of the nervous system is destroyed or damaged by malignant tissue, which is either a nervous system related malignancy or a malignant disease derived from a non-neural system); (4) infectivity; Lesions (where part of the nervous system is destroyed or damaged, for example as a result of infection by an abscess, or is associated with infection by human immunodeficiency virus, herpes zoster or herpes simplex virus, Lyme disease, tuberculosis (5) Degenerative lesions (where a portion of the nervous system is destroyed or damaged as a result of the degenerative process, including Parkinson's disease, Alzheimer's disease, Huntington's disease or the atrophic side) (Including but not limited to degeneration associated with sclerosis (ALS)); (6) associated with nutritional diseases, disorders and / or conditions Where a portion of the nervous system is destroyed or damaged by nutritional or metabolic disorders, including vitamin B12 deficiency, folic acid deficiency, Wernicke's disease, tobacco-alcohol amblyopia, Marquia furva-Vignami disease (brain (7) Primary neuropathy associated with systemic disease (diabetes (diabetic neuropathy, bell palsy), systemic lupus erythematosus, cancer or the like) Including, but not limited to, sarcoma); (8) lesions caused by toxic substances (including alcohol, lead or certain neurotoxins); and (9) demyelinating lesions, where a part of the nervous system Is destroyed or damaged by demyelinating doctrine, including multiple sclerosis, human immunodeficiency virus-related spinal cord disorders Transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and bridges but central myelinolysis include, but are not limited to).

In a preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the present invention are used to protect neurons from the damaging effects of cerebral oxygenation. According to this embodiment, the compositions of the present invention are used to treat, prevent and / or diagnose neuronal cell damage associated with cerebral oxygenation. In one aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the present invention are used to treat, prevent and / or diagnose neuronal damage associated with cerebral ischemia. In another aspect of this embodiment, the polypeptide, polynucleotide, or agonist or antagonist of the present invention treats neuronal damage associated with cerebral infarction,
Used for prevention and / or diagnosis. In another aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the present invention are used to treat, prevent and / or diagnose or prevent neuronal cell damage associated with stroke. In a further aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the present invention are used to treat, prevent and / or diagnose neuronal cell damage associated with a heart attack.

Compositions of the invention useful for treating or preventing nervous system disorders can be selected by testing for biological activity in promoting neuronal survival or differentiation. For example, but not for the purpose of limitation, compositions of the invention that elicit any of the following effects may be useful according to the invention: (1) increased survival time of neurons in the medium; (2) medium. Or increased sprouting of neurons in vivo; (3) medium or in vivo neuron-related molecules (e.g.
Increased production of choline acetyltransferase or acetylcholinesterase) for motor neurons; or (4) reduced symptoms of neuronal dysfunction in vivo. Such an effect can be measured by any method known in the art. Preferably, in a non-limiting embodiment, the increased survival time of neurons is determined by methods described herein or otherwise known in the art (eg, Arakawa et al. (J. Neurosci. 10: 3
507-3515 (1990)); and increased sprouting of neurons can be performed using methods known in the art (eg, Pest
ronk et al. (Exp. Neurol. 70: 65-82 (1980)) or B
row et al. (Ann. Rev. Neurosci. 4: 17-42 (1981)).
The increased production of neuron-related molecules can be detected using techniques known in the art and based on the molecule to be measured, bioassay, enzyme assay, antibody binding, Northern blot Motor neuron dysfunction can be measured by assessing the physical expression of motor neuron disorder (eg, weakness, motor neuron conduction velocity, or dysfunction).

In certain embodiments, motor neuron diseases, disorders and / or conditions that can be treated, prevented and / or diagnosed according to the present invention include diseases, disorders and / or conditions that may affect motor neurons and other components of the nervous system. Or state (eg,
Infarction, infection, exposure to toxins, trauma, surgical injury, degenerative disease, or malignant disease),
As well as diseases, disorders and / or conditions that selectively affect neurons (eg, amyotrophic lateral sclerosis) and include, but are not limited to, progressive spinal muscular atrophy, Progressive bulbar palsy, primary lateral sclerosis, childhood and juvenile muscular atrophy, childhood progressive bulbar paralysis (Fatio-Ronde disease), polio and post-polio symptoms, and hereditary motor and sensory neuropathy (Charcot-Marie-Tooth disease), but not limited to.

Furthermore, the polypeptides or polynucleotides of the present invention may provide neuronal survival,
It may play a role in synaptogenesis; transmission; neuronal differentiation. Accordingly, the compositions of the present invention (including polynucleotides, polypeptides, and agonists or antagonists) diagnose diseases or disorders associated with these roles, including but not limited to learning and / or cognitive impairments. And / or can be used to treat or prevent. The compositions of the present invention may be useful in the treatment or prevention of neurodegenerative disease states and / or behavioral disorders. Such neurodegenerative disease states and / or behavioral disorders include but are not limited to: Alzheimer's disease, Parkinson's disease, Huntington's disease, Tourette's syndrome,
Schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disorder,
ALS, psychosis, autism, and behavioral changes (including nutritional, sleep patterns, balance, and sensory disturbances). Furthermore, the compositions of the present invention may play a role in the treatment, prevention and / or detection of developing embryos, or developmental disorders associated with companion disorders.

In addition, the polypeptides, polynucleotides, and / or agonists or antagonists of the present invention can be used to treat neurons from diseases, injuries, disorders, or injuries associated with cerebrovascular disorders, including but not limited to: Can be useful to protect: cerebrovascular disorders (eg carotid thrombosis, carotid stenosis or moyamoya disease), amyloid angiopathy of the brain, cerebral aneurysm, cerebral hypoxia, cerebral arteriosclerosis, cerebral motion Venous malformations, cerebral artery disease, cerebral embolism and thrombosis (eg carotid artery thrombosis, sinus thrombosis and Wallenberg syndrome), epidural hematoma (eg epidural or subdural hematoma and subarachnoid hemorrhage) ), Cerebral fissure fractures, cerebral ischemia (eg,
Transient cerebral ischemia, subclavian steal syndrome, or vertebral basilar failure
basilar insufficiency)), vascular dementia (eg, multiple cerebral infarction dementia), periventricular leukomalacia, and vascular headache (eg, cluster headache).

According to a still further aspect of the invention, there is a process for utilizing a polynucleotide or polypeptide of the invention and an agonist or antagonist for therapeutic purposes, eg, to stimulate neurological cell proliferation and / or differentiation. Provided. Thus, the polynucleotides, polypeptides, agonists and / or antagonists of the present invention can be used to treat and / or detect neurological diseases. Furthermore, the polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used as markers or detection agents for certain neurological diseases or injuries.

Examples of neurological diseases that can be treated or detected using the polynucleotides, polypeptides, agonists and / or antagonists of the present invention include: brain diseases (eg, phenyls such as maternal phenylketonuria) Metabolic brain diseases, including ketonuria), pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, Wernicke brain disorder, cerebral edema, under tent (infr
atrial neoplasms including cerebellar neoplasms including neoplasms, ventricular neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms, tent epithelial neoplasms, Canavan disease, telangiectasia ataxia Cerebellar neoplasms such as cerebellar disorders such as cerebellar ataxia, including spinocerebellar degeneration, cerebellar comotor disorders, Friedreich ataxia, Machado-Joseph disease, olive bridge cerebellar atrophy, subtent neoplasm Diffuse brain sclerosis, such as diffuse periaxial encephalitis, spherical cell white matter atrophy, metachromatic leukotrophy, and subacute sclerosing panencephalitis.

Further neurological diseases that can be treated or detected using the polynucleotides, polypeptides, agonists and / or antagonists of the present invention include:
These include: cerebrovascular disorders (eg carotid artery thrombosis, carotid artery disease including carotid artery stenosis and moyamoya disease), amyloid angiopathy of the brain, cerebral aneurysm, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebrum Arterial disease, carotid thrombosis, cerebral embolism and thrombosis such as sinus thrombosis and Wallenberg syndrome, epidural hematoma, cerebral hemorrhage such as subdural hematoma and subarachnoid hemorrhage, cerebral fissure fracture, transient Cerebral ischemia, subclavian artery steal syndrome and vertebral basilar insufficiency
cerebral ischemia such as science), vascular dementia such as multiple cerebral infarction dementia, periventricular leukomalacia, vascular headache such as cluster headache.

Additional neurological diseases that can be treated or detected using the polynucleotides, polypeptides, agonists and / or antagonists of the present invention include: dementias such as AIDS dementia complex, Alzheimer's disease and Creutzfeldt- Presenile dementia such as Jakob disease, senile dementia such as Alzheimer's disease and progressive supranuclear palsy, vascular dementia such as multiple cerebral infarction dementia, encephalitis such as diffuse periaxial encephalitis, epidemic encephalitis Like encephalitis, Japanese encephalitis, viral encephalitis such as St. Louis encephalitis, tick-borne encephalitis and West Nile fever, acute disseminated encephalomyelitis, uve meningitis syndrome, post encephalitis Parkinson's disease and subsphere sclerosing panencephalitis Meningoencephalomyelitis, cerebral softening such as periventricular leukoplakia, epilepsy such as generalized epilepsy including temporomandibular spasm, and appearance Epilepsy (absence epilepsy), ME
Myoclonic epilepsy including RRF syndrome, tonic / clonic epilepsy, complex partial epilepsy, partial epilepsy such as frontal and temporal lobe epilepsy, post-traumatic epilepsy, status epilepticus such as persistent partial epilepsy, Harelfolden -Spatz syndrome.

Additional neurological diseases that can be treated or detected with the polynucleotides, polypeptides, agonists and / or antagonists of the present invention include: hydrocephalus, such as Dandy-Walker syndrome and normal pressure hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis (bulbar po l iomyelitis), cerebral pseudotumor, Rett syndrome, Reye's syndrome, thalamic diseases, cerebral toxoplasmosis, intracranial Tuberculoma and Zerweger syndrome, central nervous system infections such as AIDS dementia complex, brain abscess, subdural empyema, encephalomyelitis like equine encephalomyelitis, Venezuelan equine encephalomyelitis, necrotizing hemorrhagic Encephalomyelitis, visna, and cerebral malaria.

Additional neurological diseases that can be treated or detected using the polynucleotides, polypeptides, agonists and / or antagonists of the present invention include: meningitis such as arachnoiditis, lymphocytic choroid Aseptic meningitis such as viral meningitis including meningitis, bacterial meningitis including Haemophilus meningitis, Listeria meningitis, meningococcal characteristics such as Waterhouse-Friedersensen syndrome Meningoencephalitis, such as encephalomyelitis, pneumococcal meningitis and meningitis, fungal meningitis such as cryptocox meningitis, subdural effusion, uveal meningoencephalitis syndrome,
Myelitis such as transverse myelitis, neurosyphilis such as spinal cord fistula, polio including medullary polio and post-polio syndrome, prion diseases (eg, Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, gelstocon Stroisler syndrome, kuru, scrapie), and cerebral toxoplasmosis.

Additional neurological diseases that can be treated or detected with the polynucleotides, polypeptides, agonists and / or antagonists of the present invention include the following: Brain neoplasms, including cerebellar neoplasms, such as subtent neoplasms Canavan, a central nervous system neoplasm such as a living organism, a choroid plexus neoplasm, a ventricular neoplasm such as a hypothalamic neoplasm and a supratent neoplasm, a meningeal neoplasm, an epidural neoplasm Demyelinating diseases such as disease, diffuse cerebral sclerosis (dif), including adrenal cerebral leukodystrophies
fuse cerebral sc lerosis ), diffuse periaxial encephalitis, spherical cell leukotrophy , diffuse cerebral sclerosis of metachromatic leukotrophy , allergic encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, progressive Multifocal leukoencephalopathy, multiple sclerosis,
Bridge central myelin dissolution, transverse myelitis, optic neuritis, scrapie, lordosis, chronic fatigue syndrome, visna, high pressure Nervous syndrome (High Pressure Ne)
rvous syndrome), spinal cord diseases such as meningopathy, congenital myotonia, amyotrophic lateral sclerosis, spinal muscular atrophy such as Werdnich-Hoffmann disease, spinal cord compression, epidural neoplasm Spinal cord neoplasia, syringomyelia, spinal cord fistula, stiff man syndrome, mental retardation such as 15 q-13 minor deficiency from mother, cat cry syndrome,
De Lange syndrome, Down syndrome, Gangliosidosis such as Gangliosidosis G (M1), Zandhof disease, Tay-Sachs disease, Hartnup disease, Homocystinuria, Lawrence-Moon-Beedle syndrome, Lesch-Nyhan syndrome, Maple syrup disease, mucolipidosis such as fucose accumulation disease, neuronal ceroid sebaceous gretinosis, ocular brain kidney syndrome, phenylketonuria such as maternal phenylketonuria, Prader-Villi syndrome, Rett syndrome, Rubinstein- Nervous system failure, such as Tibi syndrome, tuberous sclerosis, WAGR syndrome, nervous system abnormalities such as total forebrain disease, incompetence including hydrangencephaly,
Spinal fusion defects such as Arnold-Chiari malformations, brain hernias, meningoceles, meningoceles, cystic spina bifida and latent spina bifida.

Additional neurological diseases that can be treated or detected using the polynucleotides, polypeptides, agonists and / or antagonists of the present invention include: hereditary motor and sensory neuron disorders, including Charcot-Marie disease, Hereditary sensations and autonomic neuropathies such as visual atrophy, Lefsum disease, hereditary spastic paraplegia, Verdwig-Hoffmann disease, congenital analgesia and familial autonomic neuropathy, neurological manifestations (e.g. Agnosia, including Gerstmann syndrome), amnesia such as retrograde amnesia, apraxia, neurogenic bladder, weakness, hearing loss,
Partial hearing deletions, loud (l ou dness) recruitment and communication disorders, such as hearing disorders, including tinnitus, Shitsushosho, name aphasia, language disorders such as aphasia including Broca aphasia and Wernicke aphasia, acute loss Dyslexia such as dyslexia, speech development disorders such as language development disorders, name aphasia, broka aphasia and aphasia including Vernicke aphasia, accumulation disorders, grammatical disorders, reverberation language, speechlessness including speechlessness and stuttering Impairment of communication, dysphonia such as ataxia and hoarseness, decerebral rigidity, delirium, fiber bundle spasm, hallucinations, meningopathy, maternal 15 q-13 microdefect, ataxia, athetosis, chorea, ataxia Dysfunction, hypoxia, hypotonia, movement disorders such as myoclonus, tics, torticollis and tremor, muscle stiffness such as stiff man syndrome Nerve hypertension, muscle spasticity, nerve palsy like facial palsy including ear shingles, gastric palsy, hemiplegia, eye muscle palsy like double vision, chronic like Duane's syndrome, Horner's syndrome, Keene's syndrome Progressive extraocular palsy, bulbar palsy, tropical convulsions paraplegia, Brown-Sekar syndrome, paraplegia such as limb paralysis, respiratory paralysis and vocal cord paralysis, paralysis, phantom limbs, taste dysfunction and taste dysfunction Visual disorders such as taste disorders such as amblyopia, blindness, blindness, color blindness, double vision, half-blindness, visual stigma and subnormal vision, hypersomnia including Kleine-Levin syndrome, insomnia and sleepwalking Sleep disorders like, spasticity like opening disorders, coma, persistent plant condition and loss of consciousness like fainting and dizziness, neuromuscular diseases like congenital myotonia, muscle atrophy Lateral sclerosis, Lambert-Eaton myasthenia syndrome, motor neuropathy, spinal muscular atrophy, Charcot-Marie disease and muscular atrophy such as Werdnich-Hoffmann disease, posterior polio syndrome, muscular dystrophy, myasthenia gravis, atrophy Myotonia, congenital myotonia, nemarin myopathy, familial limb paralysis, multiple paramyloclonus (Multiplex Paramyl)
oclonus), tropical convulsions paraplegia and stiff man syndrome, peripheral nervous system diseases such as apical pain, renal amyloidosis, Ardy syndrome, B
such as arre-Lieou syndrome, familial autonomic disorder, Horner syndrome, reflex sympathetic dystrophy and autonomic nervous system diseases such as Shy-Drager syndrome, inner ear neurological diseases such as acoustic neuroma including neurofibromatosis 2 Cranial nerve disease,
Facial nerve disease such as facial neuralgia, Mercerson-Rosenthal syndrome, oculomotor disorder including amblyopia, nystagmus, oculomotor paralysis, ocular muscular paralysis such as Duane's syndrome, chronic syndrome including Keener syndrome Extraocular muscular palsy, strabismus such as esotropia and exotropia, oculomotor palsy, ocular neurological disorders such as ocular atrophy including hereditary ocular atrophy, optic disc crystal cavity, optic nerve such as optic neuromyelitis Flames, papilledema,
Demyelinating diseases such as trigeminal neuralgia, vocal cord paralysis, optic neuromyelitis and kyphosis, diabetic neuropathy such as diabetic foot.

Additional neurological diseases that can be treated or detected using the polynucleotides, polypeptides, agonists and / or antagonists of the present invention include: nerve squeezing syndromes such as carpal tunnel syndrome, tarsal tunnel syndrome Thoracic outlet syndrome, ulnar nerve compression syndrome, causalgia, cervical neuralgia,
Neuralgia such as facial and trigeminal neuralgia, experimental allergic neuritis, ophthalmic neuritis, polyneuritis, polyneuropathy and radiculitis (eg, polyneuropathy, hereditary motor and sensation) Neuropathies (eg, Charcot-Marie disease, hereditary ocular atrophy, refsum disease, hereditary spastic paraplegia and Werdnich-Hoffmann disease, hereditary sensory and autonomic neuropathies (congenital analgesia and familial autonomic neuropathy) Neuritis such as POEMS syndrome, sciatica, gustatory sweat syndrome and tetany)).

(Infectious diseases)
The polynucleotides or polypeptides, and / or agonists or antagonists of the present invention can be used to treat, prevent and / or diagnose infectious agents. For example, infectious diseases can be treated, prevented and / or diagnosed by raising the immune response, in particular by increasing proliferation and differentiation of B cells and / or T cells. The immune response can be raised by either raising an existing immune response or initiating a new immune response. Alternatively, the polynucleotide or polypeptide of the invention and / or agonist or antagonist also does not necessarily elicit an immune response,
Can directly inhibit infectious agents.

A virus is an example of an infectious agent that can cause a disease or condition that can be treated, prevented, and / or diagnosed by a polynucleotide or polypeptide, and / or agonist or antagonist of the present invention. Examples of viruses include, but are not limited to, the following DNA and RNA viruses and viridae: arbovirus, adenoviridae, arenaviridae,
Arterivirus, Birnaviridae, Bunyaviridae, Calciviridae,
Sarcoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (hepatitis), Herpesviridae (eg, cytomegalovirus, herpes simplex, herpes zoster), mononegavirus (Mononegavirus) (eg, Paramyxoviridae, Measles virus, Rhabdoviridae), Orthomyxoviridae (eg, Influenza A, Influenza B, and Parainfluenza), papillomavirus, Papovaviridae, Parvoviridae, Picornavirus Family,
Poxviridae (eg, pressure ulcer or vaccinia), Reoviridae (eg, rotavirus), Retroviridae (HTLV-I, HTLV-II, lentivirus), and Togaviridae (eg, Rubivirus genus). Viruses that fall within these families can cause a variety of diseases or conditions including, but not limited to: arthritis, bronchiolitis, respiratory syncytial virus, encephalitis, ocular infections (eg, conjunctivitis) , Keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, chronic activity, Delta), Japanese encephalitis B, Argentine hemorrhagic fever, Chingnia, Rift Valley fever, yellow fever, meninges flame,
Opportunistic infections (eg AIDS), pneumonia, Burkitt lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, cold, polio, leukemia, rubella, sexually transmitted diseases, skin diseases (eg , Capoji, warts), and viremia.
Any of these conditions or diseases can be treated, prevented and / or diagnosed using the polypeptides or polynucleotides, or agonists or antagonists of the present invention. In certain embodiments, the polynucleotides, polypeptides, or agonists or antagonists of the present invention are used for the following treatments, preventions and / or diagnoses: meningitis, dengue fever, EBV, and / or hepatitis (eg, Hepatitis B). In further specific embodiments, the polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients who are non-responsive to one or more other commercially available hepatitis vaccines. In a more specific embodiment, the polynucleotides, polypeptides, or agonists or antagonists of the present invention are used to treat, prevent and / or diagnose AIDS.

Similarly, bacterial or fungal factors that can cause disease or symptoms and that can be treated, prevented and / or diagnosed by the polynucleotides or polypeptides, and / or agonists or antagonists of the present invention include the following Gram negative and Includes, but is not limited to, Gram-positive bacteria and bacteriophages and fungi: Actinomycetals (eg Corynebac
terium, Mycobacterium, Norcardia), Cryp
tococcus neoformans, Aspergillosis, Ba
Cillaceae (eg, Anthrax, Clostridium), B
Acteoidaceae, Blastomycosis, Bordetel
la, Borrelia (eg, Borrelia burgdorferi
), Brucellosis, Candidiasis, Campylobac
ter, Coccidiomycosis, Cryptococcosi
s, Dermatocytoses, E.M. coli (for example, Enteroto
xigenic E.M. E. coli and Enterohemorrhagic E
. coli), Enterobacteriaceae (Klebsiella
, Salmonella (eg, Salmonella typhi, and Salmonella paratyphi), Serratia, Yersi
nia), Erysipelotrix, Helicobacter, Leg
ionellosis, Leptospirosis, Listeria, My
coplasma materials, Mycobacterium leprae, V
ibrio cholerae, Neisseriaceae (for example, Aci
netobacter, Gonorrhea, Menigococcal), M
eisseria meningitidis, Pasteurellacea
Infectious diseases (e.g., Actinobacillus, Hemophilus (
For example, Hemophilus influenza B), Pasteurell
a), Pseudomonas, Rickettsiaceae, Chlamy
diaceae, Syphilis, Shigella spp. , Staph
Yelococcal, Meningiococcal, Pneumococca
l as well as Streptococcal (eg Streptococcus
pneumoniae and group B Streptococcus). These bacterial or fungal families can cause diseases or symptoms including but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic Infections (eg, infections associated with AIDS), peritonitis, prosthesis-related infections, Reiter's disease, respiratory tract infections (eg pertussis or empyema), sepsis, Lyme disease, cat scratch disease, dysentery, paratyphoid fever, Food poisoning, typhoid fever, pneumonia, gonorrhea, meningitis (eg meningitis type A and B), chlamydia, syphilis, diphtheria, lei disease, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo , Rheumatic fever, scarlet fever, sexually transmitted diseases, skin diseases (eg cellulitis, dermatomycosis (de
rmatocycles)), toxemia, urinary tract infection, wound infection. The polypeptides or polynucleotides, agonists or antagonists of the present invention can be used to treat, prevent and / or diagnose any of these symptoms or diseases. In certain embodiments, the polynucleotides, polypeptides, agonists or antagonists of the present invention are used to treat, prevent and / or diagnose the following: tetanus, diphtheria, botulinum, and / or type B meningitis .

Further, diseases or conditions caused by parasitic factors that can be treated, prevented and / or diagnosed by the polynucleotides or polypeptides and / or agonists or antagonists of the present invention include the following families or classes: Without limitation: amebiasis, babesiosis, coccidiosis, cryptosporidiosis, binuclear amebiasis (Dientamoebi)
asis), epidemics, ectoparasites, giardia flagellate disease, helminthiasis, leishmaniasis, theileriasis, toxoplasmosis, trypanosomiasis, and Trichomonas disease, and Sporozoan
(For example, Plasmodium virax, Plasmodium fal
ciparium, Plasmodium malariae and Plasm
odium oval). These parasites can cause a variety of diseases or conditions including, but not limited to: scabies, tsutsugamushi disease, ocular infections, intestinal diseases (eg, dysentery, Giardia flagellate disease), liver diseases, lungs Diseases, opportunistic infections (eg AIDS related), malaria, pregnancy complications, and toxoplasmosis. Any of these symptoms or diseases may be treated, prevented and / or diagnosed using the polynucleotides or polypeptides, or agonists or antagonists of the present invention. In certain embodiments, the polynucleotide of the invention,
The polypeptide, or agonist or antagonist, treats malaria,
Used for prevention and / or diagnosis.

Preferably, treatment or prophylaxis with the polynucleotides or polypeptides of the invention and / or agonists or antagonists involves administering to the patient an effective amount of the polypeptide or removing cells from the patient and Either by supplying nucleotides to the cells and returning the engineered cells back to the patient (ex vivo treatment). Furthermore, the polypeptides or polynucleotides of the present invention can be used as antigens in vaccines to elicit an immune response against infectious diseases.

(Regeneration)
The polynucleotides or polypeptides and / or agonists or antagonists of the present invention can be used to differentiate, proliferate, and attract cells leading to tissue regeneration (see Science 276: 59-87 (1997). ) Using tissue regeneration, congenital defects, trauma (wounds, burns, incisions, or ulcers), aging, diseases (eg, osteoporosis, osteoarthritis (osteocar
thrisis), periodontal disease, liver failure), surgery including cosmetic surgery, fibrosis, reperfusion injury, or tissue damaged by systemic cytokine damage may be repaired, replaced, or protected.

Tissues that can be regenerated using the present invention include: organs (eg,
Pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle, or heart muscle),
Vascular system (including blood vessels and lymphatics), nerves, hematopoiesis, and skeletal (bone, cartilage, tendon, and ligament) tissues. Preferably, regeneration occurs with no scarring or reduced scarring. Regeneration can also include angiogenesis.

Furthermore, the polynucleotides or polypeptides and / or agonists or antagonists of the present invention may increase the regeneration of tissues that are difficult to heal. For example, by increasing tendon / ligament regeneration, recovery time after injury is accelerated. The polynucleotides or polypeptides, and / or agonists or antagonists of the present invention can also be used prophylactically in an attempt to avoid damage. Particular diseases that can be treated, prevented and / or diagnosed include tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. Additional examples of tissue regeneration of non-healing wounds include pressure ulcers, vascular failure, surgical wounds, and ulcers associated with traumatic wounds.

Similarly, nerve and brain tissue can also be regenerated by using the polynucleotides or polypeptides and / or agonists or antagonists of the present invention to grow and differentiate nerve cells. Treatment using this method,
Diseases that can be prevented and / or diagnosed include central and peripheral nervous system diseases, neurological disorders, or mechanical and traumatic diseases, disorders and / or conditions (eg spinal cord disorders, head trauma, cerebrovascular Disease, and stroke). Specifically, diseases associated with peripheral nerve injury, peripheral neuropathy (eg, resulting from chemotherapy or other medical therapy), localized neuropathy, and central nervous system diseases (eg, Alzheimer's disease, Parkinson's disease, Huntington) Disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome) can all be treated, prevented and / or diagnosed using the polynucleotides or polypeptides and / or agonists or antagonists of the present invention.

(Chemotaxis)
The polynucleotides or polypeptides and / or agonists or antagonists of the present invention may have chemotactic activity. Chemotactic molecules can direct cells (eg, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells and / or endothelial cells) to specific sites in the body (eg, Attract or mobilize (sites of inflammation, infection, or hyperproliferation). The recruited cells can then repel and / or heal certain trauma or abnormalities.

The polynucleotides or polypeptides and / or agonists or antagonists of the present invention may increase the chemotactic activity of certain cells. These chemotactic molecules are then used to increase the number of cells targeted to a specific location in the body, thereby causing inflammation, infection, hyperproliferative disease, disorder and / or condition, or any May be treated, prevented and / or diagnosed. For example, chemotactic molecules can be used to treat, prevent and / or diagnose wounds and other trauma to tissues by attracting immune cells to the injured location. The chemotactic molecules of the present invention can also attract fibroblasts, which treat, prevent and / or treat wounds.
Or it can be used to diagnose.

It is also contemplated that the polynucleotides or polypeptides and / or agonists or antagonists of the present invention may inhibit chemotactic activity. These molecules can also be used to treat, prevent and / or diagnose diseases, disorders and / or conditions. Thus, the polynucleotides or polypeptides and / or agonists or antagonists of the present invention can be used as chemotactic inhibitors.

(Binding activity)
The polypeptides of the invention can be used to screen for molecules that bind to, or molecules that bind to the polypeptide. Binding of the polypeptide to the molecule can activate (agonist), increase, inhibit (antagonist), or decrease the activity of the bound polypeptide or molecule.
Examples of such molecules include antibodies, oligonucleotides, proteins (eg, receptors), or small molecules.

Preferably, the molecule is closely related to a natural ligand (eg, a fragment of a ligand) of the polypeptide, or a natural substrate, ligand, structural mimetic, or functional mimetic (Coligan et al., Current Prot
ocols in Immunology 1 (2): see Chapter 5 (1991)). Similarly, the molecule may be closely related to the natural receptor to which the polypeptide binds, or at least to a fragment of the receptor that can be bound by the polypeptide (eg, the active site). In any case, the molecule can be rationally designed using known techniques.

Preferably, screening for these molecules involves producing appropriate cells that express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include mammals, yeast and Drosoph.
ila, or E.I. cells derived from E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably observed to observe binding, stimulation of activity, or inhibition of activity of either the polypeptide or the molecule. Is contacted with a test compound potentially containing

The assay may simply test the binding of the candidate compound to the polypeptide, where binding is detected by a label or in an assay for competition with a labeled competitor. In addition, the assay may test whether the candidate compound produces a signal produced by binding to the polypeptide.

Alternatively, the assay can be performed using cell-free preparations, polypeptides / molecules attached to a solid support, chemical libraries, or natural product mixtures. The assay also simplifies the steps of mixing a candidate compound with a solution containing the polypeptide, measuring the activity / binding of the polypeptide / molecule, and comparing the activity / binding of the polypeptide / molecule to a standard. Can be included.

Preferably, an ELISA assay can measure the level or activity of a polypeptide in a sample (eg, a biological sample) using a monoclonal or polyclonal antibody. An antibody can either bind directly or indirectly to a polypeptide, or compete with the polypeptide for a substrate,
The level or activity of the polypeptide can be measured.

In addition, the receptors to which the polypeptides of the present invention bind can be obtained in a number of ways known to those skilled in the art (eg, ligand panning and FACS sorting (Coliga).
n et al., Current Protocols in Immun. , 1 (2),
Chapter 5 (1991)). For example, expression cloning is used when polyadenylated RNA is prepared from cells that respond to the polypeptide, such as NIH3T3 cells (which are known to contain multiple receptors for FGF family proteins), And SC-3 cells, and cDNA libraries made from this RNA, are divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptide. Transfected cells growing on glass slides are exposed to the polypeptides of the invention after labeling them. The polypeptide can be labeled by a variety of means, including iodination or encapsulation of a recognition site for a site-specific protein kinase.

After immobilization and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and subpools are prepared and retransfected using an iterative subpooling and rescreening process to ultimately yield a single clone encoding the putative receptor.

As an alternative approach for receptor identification, labeled polypeptides can link or extract a preparation that expresses the receptor molecule in photoaffinity with the cell membrane. Cross-linked material is separated by PAGE analysis and exposed to X-ray film. A labeled complex comprising a receptor for the polypeptide can be excised;
It can be separated into peptide fragments and subjected to protein microsequencing. Using the amino acid sequence obtained from microsequencing, a set of degenerate oligonucleotide probes is designed to screen a cDNA library and identify genes encoding putative receptors.

In addition, gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling techniques (collectively referred to as “DNA shuffling”) can be used to modulate the activity of the polypeptides of the present invention. Effectively produce polypeptide agonists and antagonists. In general, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,835.
7,458, and Patten, P .; A. Et al., Curr. Opinion
Biotechnol. 8: 724-33 (1997); Harayama,
S. Trends Biotechnol. 16 (2): 76-82 (1998)
Hansson, L .; O. Et al. Mol. Biol. 287: 265-7
6 (1999); and Lorenzo, M .; M.M. And Blasco, R .;
See Biotechniques 24 (2): 308-13 (1998), each of these patents and publications incorporated herein by reference. In one embodiment, alteration of the polynucleotides and corresponding polypeptides of the invention can be accomplished by DNA shuffling. DNA shuffling involves two or more DNAs by homologous recombination or site-specific recombination.
Includes the assembly of segments into the desired polynucleotide sequence of the molecules of the invention. In another embodiment, the polynucleotides of the invention and corresponding polypeptides are subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination, Can be changed. In another embodiment, one or more components of a polypeptide of the invention,
Motifs, sections, portions, domains, fragments, etc. can be recombined with one or more components, motifs, sections, portions, domains, fragments, etc. of one or more heterologous molecules. In a preferred embodiment, the heterologous molecule is a family member. In a further preferred embodiment, the heterologous molecule comprises, for example, platelet derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF) -α, epidermal growth factor (EGF), fibroblast Cell growth factor (FGF), TGF-β, bone morphogenetic protein (BMP) -2, BMP-4, BM
P-5, BMP-6, BMP-7, activin A and activin B, decapentaplegic (dpp), 60A, OP-
2, dosalin, growth differentiation factor (GDF), nodule
al), MIS, inhibin-α, TGF-β1, TGF-β2, TGF-β3
, TGF-β5, and growth factors such as glial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of the polypeptides of the invention. Biologically active fragments are those that exhibit activity similar to, but not necessarily identical to, the activity of the polypeptide. The biological activity of the fragment may include an improved desired activity or a reduced undesirable activity.

Furthermore, the present invention provides methods for screening compounds to identify compounds that modulate the action of the polypeptides of the present invention. An example of such an assay involves combining mammalian fibroblasts, a polypeptide of the invention, a compound to be screened and ³ [H] thymidine under cell culture conditions in which fibroblasts normally grow. . The control assay can be performed in the absence of the compound to be screened and the amount of ³ [H] thymidine incorporation in each case compared to the amount of fibroblast proliferation in the presence of this compound. By determination, it can be determined whether the compound stimulates proliferation. The amount of fibroblast proliferation is ³ [H
] Measured by liquid scintillation chromatography to measure thymidine incorporation. Both agonist and antagonist compounds can be identified by this procedure.

In another method, a mammalian cell or membrane preparation that expresses a receptor for a polypeptide of the invention is incubated with the labeled polypeptide of the invention in the presence of the compound. The ability of the compound to enhance or block the interaction can then be measured. Alternatively, a known second messenger system response and receptor according to the interaction of the compound to be screened is measured, and the ability of the compound to bind to the receptor and elicit a second messenger response is determined. Determine if it is a potential agonist or antagonist. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognostic markers. Molecules discovered using these assays treat, prevent and / or diagnose diseases by activating or inhibiting polypeptides / molecules or have specific results (eg, vascular proliferation) in patients. Can be used to bring. In addition, the assay may discover factors that can inhibit or enhance the production of a polypeptide of the invention from appropriately engineered cells or tissues. Accordingly, the present invention includes a method of identifying a compound that binds to a polypeptide of the present invention comprising the following steps: (a) incubating a candidate binding compound with the polypeptide; and (b) has binding occurred? Determining whether or not. Furthermore, the invention includes a method of identifying an agonist / antagonist comprising the following steps: (a) incubating a candidate compound with a polypeptide, (b) assaying biological activity, and (b) ) Determining whether the biological activity of the polypeptide has been altered.

Further, by using the β-pleated sheet region contained in the polypeptide sequence of the protein, a molecule that binds the polypeptide of the present invention can be identified experimentally. Thus, certain embodiments of the invention include the amino acid sequence of each β-pleated sheet region in the disclosed polypeptide sequence, or the amino acid sequence of each β-pleated sheet region in the disclosed polypeptide sequence And a polynucleotide encoding a polypeptide. Further embodiments of the invention encode a polypeptide comprising any combination or all contained in, or consisting of, any combination or all contained in the polypeptide sequence of the invention. To a polynucleotide. Further preferred embodiments of the present invention comprise the amino acid sequence of each of the β-pleated sheet regions in one of the polypeptide sequences of the present invention, or β in one of the polypeptide sequences of the present invention.
The present invention relates to a polypeptide consisting of the amino acid sequence of each pleated sheet region. Further embodiments of the invention include any combination or all of the β-pleated sheet regions in one of the polypeptide sequences of the invention, or any of the β-pleated sheet regions in one of the polypeptide sequences of the invention. It relates to a polypeptide comprising a combination or all.

(Targeted delivery)
In another embodiment, the present invention provides a method of delivering a composition to a targeted cell that expresses a receptor for a polypeptide of the invention, or a cell that expresses a cell-bound form of the polypeptide of the invention. .

As discussed herein, a polypeptide or antibody of the present invention has a hydrophobic, hydrophilic, ionic and / or covalent interaction with a heterologous polypeptide, heterologous nucleic acid, toxin or prodrug. Can meet through. In one embodiment, the invention provides a polypeptide of the invention associated with a heterologous polypeptide or nucleic acid (
A method is provided for specific delivery of a composition of the invention to a cell by administering an antibody). In one example, the present invention provides a method for delivering a therapeutic protein into a targeted cell. In another example, the invention can be a single-stranded nucleic acid (eg, antisense or ribozyme) or a double-stranded nucleic acid (eg, can be integrated into the genome of a cell, or can be replicated episomally and transcribed. , DNA) is provided for delivery to targeted cells.

In another embodiment, the invention provides for the specific destruction of cells by administering a polypeptide of the invention associated with a toxin or cytotoxic prodrug (eg, a polypeptide of the invention or an antibody of the invention) ( For example, a method for destruction of tumor cells is provided.

"Toxins" refers to endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or cells or cell surfaces that cause cell death under defined conditions Means a compound that binds and activates any molecule or enzyme not normally present. Toxins that can be used in accordance with the methods of the present invention include, but are not limited to: Radioisotopes known in the art, compounds that bind intrinsic or induced endogenous cytotoxic effector systems (Eg, antibody (or complement fixation including part thereof), thymidine kinase, endonuclease, RNAse, alpha toxin, ricin,
Abrin, Pseudomonas endotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. "
By “cytotoxic prodrug” is meant a non-toxic compound that is converted to a cytotoxic compound by an enzyme that is normally present in the cell. Cytotoxic prodrugs that can be used according to the methods of the invention include glutamyl derivatives of benzoic acid mustard alkylating agents, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubicin, and phenoxyacetamide derivatives of doxorubicin. However, it is not limited to these.

(Drug screening)
Further contemplated is the use of the polypeptides of the invention, or polynucleotides encoding these polypeptides, for screening for molecules that alter the activity of the polypeptides of the invention. Such methods include contacting a polypeptide of the invention with a selected compound suspected of having antagonistic or agonistic activity, and assaying the activity of these polypeptides following binding. To do.

The invention is particularly useful for screening therapeutic compounds by using the polypeptides of the invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment used in such tests can be immobilized on a solid support, expressed on the cell surface, free in solution, or localized within the cell. One method of drug screening utilizes eukaryotic or prokaryotic host cells that are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. For example, the formation of a complex between the agent being tested and the polypeptide of the invention can be measured.

Thus, the present invention provides a screening method for drugs or any other agent that affects the activity mediated by the polypeptides of the present invention. These methods involve contacting such an agent with a polypeptide of the invention or a fragment thereof by methods well known in the art, and the presence of a complex between the agent and the polypeptide or a fragment thereof. Including assaying. In such competitive binding assays, the agent to be screened is typically labeled. After incubation, free drug is separated from the drug present in the bound form, and the amount of free or unconjugated label is a measure of the ability of a particular drug to bind to a polypeptide of the invention. .

Another technique for drug screening provides high-throughput screening for compounds with appropriate binding affinity for the polypeptides of the invention, and European Patent Application 84/03564 (published September 13, 1984) (which is , Which is incorporated herein by reference) in great detail. Briefly, a large number of different small peptide test compounds are synthesized on a solid substrate (eg, a plastic pin or some other surface). Peptide test compounds
React with the polypeptide of the present invention and wash. The bound polypeptide is then detected by methods well known in the art. The purified polypeptide is encoded directly on the plate for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

The present invention also contemplates the use of competitive drug screening assays, wherein neutralizing antibodies capable of binding a polypeptide of the invention specifically compete with a test compound for binding to the polypeptide or fragment thereof. In this style,
Antibodies are used to detect the presence of any peptide that shares one or more antigenic epitopes with a polypeptide of the invention.

(Peptides and other molecules that bind to the polypeptides of the invention)
The invention also includes screening methods for identifying polypeptides and non-polypeptides that bind to the polypeptides of the invention, as well as molecules that bind to the polypeptides of the invention identified thereby. These binding molecules are useful, for example, as agonists and antagonists of the polypeptides of the present invention. Such agonists and antagonists may be used in accordance with the present invention in the therapeutic embodiments described in detail below.

This method includes the following steps:
a. Contacting a polypeptide of the invention with multiple molecules; and b. Identifying a molecule that binds to a polypeptide of the invention.

The step of contacting a polypeptide of the invention with multiple molecules can be effected by a number of methods. For example, one skilled in the art can consider immobilizing a polypeptide of the invention on a solid support and contacting a solution of multiple molecules with the immobilized polypeptide of the invention. Such a procedure is similar to an affinity chromatography process using an affinity matrix composed of immobilized polypeptides of the invention. Molecules with selective affinity for the polypeptides of the invention can then be purified by affinity selection. The nature of the solid support, the process for attaching the polypeptide of the invention to this solid support, the solvent, and the conditions for affinity isolation or affinity selection are largely conventional and well known to those skilled in the art. It is.

Alternatively, multiple polypeptides can also be separated into substantially separate fractions containing a subset of polypeptides or individual polypeptides. For example, a large number of polypeptides can be separated by methods known to those skilled in the art regarding separation of polypeptides such as gel electrophoresis, column chromatography and the like. Individual polypeptides can also be produced by transformed host cells (eg, recombinant phage) in such a way that they are expressed on or near the outer surface of the host cell. Individual isolates can then be “probed” with the polypeptides of the invention, and in the presence of inducers that would be required for expression as needed, It is determined whether any selective affinity interaction has occurred with the clone. Prior to contacting the polypeptides of the present invention with each fraction containing individual polypeptides, these polypeptides can first be transferred to a solid support for further convenience. Such a solid support may simply be a piece of filter membrane made, for example, of nitrocellulose or nylon. In this manner, positive clones can be identified from a population of expression libraries of transformed host cells that have a DNA construct that encodes a polypeptide having selective affinity for a polypeptide of the invention. . Furthermore, the amino acid sequence of a polypeptide having selective affinity for the polypeptide of the present invention can be determined directly by conventional means, or the coding sequence of the DNA encoding this polypeptide is often more and more convenient. Can be determined. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid sequence is determined from the polypeptide itself, microsequencing techniques can be used. This sequencing technique can include mass spectrometry.

In certain circumstances, before attempting to determine or detect the presence of a selective affinity interaction, either the unbound polypeptide of the invention or the unbound polypeptide is treated with a polypeptide of the invention and a number of polypeptides. It may be desirable to wash away from the peptide mixture. Such a washing step may be particularly desirable when the polypeptide or multiple polypeptides of the invention are bound to a solid support.

A large number of molecules provided in accordance with the present methods can be used as diversity libraries (eg, random or combinatorial peptide or non-peptide libraries that can screen for molecules that specifically bind to the polypeptides of the invention). Can be provided. Many libraries that can be used (eg, chemical synthesis libraries, recombinant libraries (eg, phage display libraries),
And in vitro translation-based libraries) are known in the art. Examples of chemical synthesis libraries are described below: Fodor et al., 1991, Sci.
ence 251: 767-773; Houghten et al., 1991, Natu
re 354: 84-86; Lam et al., 1991, Nature 354: 82.
-84; Medynski, 1994, Bio / Technology 12:
709-710; Gallop et al., 1994, J. MoI. Medicinal Che
history 37 (9): 1233-1251; Ohlmyer et al., 199
3, Proc. Natl. Acad. Sci. USA 90: 10922-10
926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA
91: 11142-11426; Houghten et al., 1992, Biote.
chniques 13: 412; Jayawickreme et al., 1994, P
roc. Natl. Acad. Sci. USA 91: 1614-1618; S
almon et al., 1993, Proc. Natl. Acad. Sci. USA 9
0: 11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad
. Sci. USA 89: 5381-5383.

Examples of phage display libraries are described below: Scott and Smith, 1990, Science 249: 386-390; Dev
lin et al., 1990, Science, 249: 404-406; Christ.
ian, R.A. B. Et al., 1992, J. Am. Mol. Biol. 227: 711-71
8); Lenstra, 1992, J. MoI. Immunol. Meth. 152: 1
49-157; Kay et al., 1993, Gene 128: 59-65; and PCT Publication No. WO 94/18318 (August 18, 1994).

In vitro translation-based libraries include the following: PCT publication number WO9
1/05058 (April 18, 1991); and Mattheakis et al., 1
994, Proc. Natl. Acad. Sci. USA 91: 9022-9
Although what is described in 026 is mentioned, it is not limited to these.

Examples of non-peptide libraries include benzodiazepine libraries (eg, Bunin et al., 1994, Proc. Natl. Acad. Sci. USA).
91: 4708-4712) can be adapted for use. Peptoid library (Simon et al., 1992, Proc. Natl. Acad
. Sci. USA 89: 9367-9371) can also be used. Another example of a library that can be used is Ostresh et al. (1994, Proc. Nat.
l. Acad. Sci. USA 91: 11138-11142), where the amide function in the peptide is over-methylated (permet).
hydrated) to generate a chemically transformed combinatorial library.

The various non-peptide libraries that are useful in the present invention are large. For example, Ecker and Crooke (1995, Bio / Technology).
13: 351-360) include benzodiazepines, hydantoins, piperazinediones, biphenyls, sugar analogs, β-mercaptoketones, arylacetic acids, acylpiperazines, benzopyrans, cubans cubane), xanthines, amine imides and oxazolones.

Non-peptide libraries can be broadly classified into two types: modified monomers and oligomers. The modified monomer library uses a relatively simple skeletal structure on which various functional groups are added. Often the scaffold is a molecule with known useful pharmacological activity. For example, the skeleton can be a benzodiazepine structure.

Non-peptide oligomer libraries use a large number of monomers that are assembled together in a manner that creates a new shape depending on the order of the monomers. Among the monomer units used are carbamate, pyrrolinone.
) And morpholino. Peptoids (peptide-like oligomers whose side chains are attached to the alpha amino group rather than the alpha carbon) form the basis of another version of a non-peptide oligomer library. The first non-peptide oligomer library uses a single type of monomer and thus contains a repetitive backbone. Modern libraries use more than one monomer and give a library with added degrees of freedom.

Screening the library can be accomplished by any of a variety of commonly known methods. For example, the following references disclosing the screening of peptide libraries: Palmley and Smith, 1989, Adv.
. Exp. Med. Biol. 251: 215-218; Scott and Sm
ith, 1990, Science 249: 386-390; Fowlkes
1992; BioTechniques 13: 422-427; Olde
nburg et al., 1992, Proc. Natl. Acad. Sci. USA 8
9: 5393-5397; Yu et al., 1994, Cell 76: 933-945.
Staudt et al., 1988, Science 241: 577-580; Bo
ck et al., 1992, Nature 355: 564-566; Tuerk et al., 1
992, Proc. Natl. Acad. Sci. USA 89: 6988-6
992; Ellington et al., 1992, Nature 355: 850-8.
52; US Pat. No. 5,096,815, US Pat. No. 5,223,409, and US Pat. No. 5,198,346 (all to Ladner et al.); Reb
See ar and Pabo, 1993, Science 263: 671-673; and PCT Publication No. WO 94/18318.

In certain embodiments, the screening to identify molecules that bind a polypeptide of the invention comprises contacting a member of the library with a polypeptide of the invention immobilized on a solid phase, and the polypeptide of the invention Can be performed by collecting these libraries that bind to An example of such a screening method, referred to as “panning” is, for example, Parmley and Sm.
ith, 1988, Gene 73: 305-318; Fowlkes et al., 19
92, BioTechniques 13: 422-427; PCT publication number W
O94 / 18318; as well as references cited therein.

In another embodiment, a two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature).
340: 245-246; Chien et al., 1991, Proc. Natl. Ac
ad. Sci. USA 88: 9578-9582) can be used to identify molecules that specifically bind to a polypeptide of the invention.

When the polypeptide of the present invention bound to a molecule is a polypeptide, the polypeptide can be any peptide library (random peptide library,
From a combinatorial peptide library or a bias peptide library). The term “biased” means that in the peptide in this case, the method of generating the library is manipulated to limit one or more parameters that govern the diversity of molecular collection that occurs. As used herein.

Thus, a truly random peptide library creates a collection of peptides where the probability of finding a particular amino acid at a given position in the peptide is the same for all 20 amino acids. However, for example, by specifying that lysine occurs at every 5th amino acid or fixing and specifying that the 4th, 8th and 9th positions of the decapeptide library contain only arginine, the bias is live. Can be introduced into the rally. Obviously, many types of bias can be contemplated and the invention is not limited to any particular bias. In addition, the present invention contemplates certain types of peptide libraries (eg, libraries that use a phage display peptide library and a DNA construct comprising a lambda phage vector with a DNA insert).

As described above, a molecule (which is a polypeptide) that binds to the polypeptide of the present invention.
The polypeptide has from about 6 to less than about 60 amino acid residues;
Preferably from about 6 to about 10 amino acid residues, and more preferably from about 6 to about 22 amino acids. In another embodiment, a polypeptide that binds to a polypeptide of the invention has an amino acid range of 15-100, or an amino acid range of 20-50.

Selected polypeptides of the present invention linked to a molecule can be obtained by chemical synthesis or recombinant expression.

(Antisense and ribozyme (antagonist))
In a particular embodiment, the antagonist according to the invention is a nucleic acid corresponding to the sequence contained in SEQ ID NO: X or its complementary strand and / or the nucleotide sequence contained in the deposited clone. In one embodiment, the antisense sequence is generated internally by the organism, and in another embodiment, the antisense sequence is administered separately (eg, O'Connor, Neuroch).
em. 56: 560 (1991)). Oligodeoxyynuc
leotides as Antisense Inhibitors of
Gene Expression, CRC Press, Boca Raton
, FL (1988). Antisense technology can be used to control gene expression through antisense DNA or RNA or through triple helix formation. Antisense technology is described, for example, in Okano, Neurochem. 56:56
0 (1991); Oligodeoxynucleotides as Ant
isense Inhibitors of Gene Expression
, CRC Press, Boca Raton, FL (1988). Triple helix formation is described, for example, by Lee et al., Nucleic Acids Res.
search 6: 3073 (1979); Cooney et al., Science, 2
41: 456 (1988); and Dervan et al., Science 251:
1300 (1991). These methods are based on the binding of polynucleotides to complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of non-lymphocytic leukemia cell line HL-60 and other cell lines has been described previously (Wickstrom et al. (1988); Anfossi et al. (198
9)). These experiments were performed in vitro by incubating the cells with oligoribonucleotides. A similar procedure for in vivo applications is
It is described in WO91 / 15580. Briefly, an oligonucleotide pair for a given antisense RNA is generated as follows: a sequence complementary to the first 15 bases of the open reading frame, a 5-terminal EcoRI site and a 3-terminal HindIII Adjacent to the site. The oligonucleotide pair was then heated at 90 ° C. for 1 minute and then 2 × ligation buffer (20 mM TRIS HC
l pH 7.5, 10 mM MgCl ₂ , 10 mM dithiothreitol (DT
T) and 0.2 mM ATP) and then ligated to the EcoR1 / HindIII site of the retroviral vector PMV7 (WO91
/ 15580).

For example, an antisense RNA oligonucleotide of about 10-40 base pairs in length can be designed using the 5 ′ coding portion of a polynucleotide encoding the mature polypeptide of the invention. DNA oligonucleotides are designed to be complementary to regions of the gene involved in transcription, thereby inhibiting transcription and receptor production. Antisense RNA oligonucleotides hybridize to mRNA in vivo and block translation of mRNA molecules into receptor polypeptides.

In one embodiment, the antisense nucleic acid of the invention is produced intracellularly by transcription from a foreign sequence. For example, the vector or a portion thereof is transcribed to produce the antisense nucleic acid (RNA) of the present invention. Such vectors contain a sequence that encodes an antisense nucleic acid of the invention. Such a vector can retain the episome or be chromosomally integrated so long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. The vector may be a plasmid, virus, etc. known in the art used for replication and expression in vertebrate cells. Expression of the sequence encoding a polypeptide of the invention or a fragment thereof is obtained by any promoter known in the art that acts in vertebrates, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include the SV40 early promoter region (Bernist and Chamber, Nature, 29: 304-3).
10 (1981)), a promoter contained in the 3 ′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22: 787-797 (1980)),
Herpes thymidine promoter (Wagner et al., Proc. Natl. Aca
d. Sci. U. S. A. 78: 1441-1445 (1981)), regulatory sequences of the metallothionein gene (Brinster et al., Nature, 296: 39).
-42 (1982)), but is not limited thereto.

The antisense nucleic acid of the present invention contains a sequence complementary to at least a part of the RNA transcript of the gene of interest. However, complete complementarity is preferred but not necessary. As used herein, a sequence “complementary to at least a portion of RNA” means a sequence that has sufficient complementarity to be able to hybridize to RNA and forms a stable duplex; Thus, in the case of the double-stranded antisense nucleic acids of the invention, a single strand of double stranded DNA can be tested or triplex formation can be assayed.
The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid. In general, longer nucleic acids that hybridize may contain more base mismatches with the RNA sequences of the present invention, which may contain stable duplexes (or may be triplexed), And still can be formed. One skilled in the art can ascertain the degree of mismatch tolerance by using standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5 ′ end of the message (eg, 5 ′ untranslated sequences up to and including the AUG start codon) should work most efficiently in inhibiting translation. However, sequences complementary to the 3 ′ untranslated sequence of mRNA have also been shown to be effective in inhibiting translation of mRNA. See generally Wagner, R .; Nature 372: 333-335 (199
See 4). Thus, 5′- or 3 ′ of the polynucleotide sequences of the invention
An oligonucleotide complementary to any of the non-translated non-coding regions of
It can be used in antisense approaches that inhibit RNA translation. mRNA 5
'An oligonucleotide complementary to the untranslated region should contain the complement of the AUG start codon. Antisense oligonucleotides complementary to the mRNA coding region are less efficient inhibitors of translation, but can be used in accordance with the present invention. Whether designed to hybridize to the 5 ′ region, 3 ′ region or coding region of an mRNA, antisense nucleic acids should be at least 6 nucleotides in length and preferably 6 to about 50 An oligonucleotide spanning the nucleotide length. In certain aspects, the oligonucleotide has at least 10 nucleotides, at least 17 nucleotides, at least 25
Nucleotides or at least 50 nucleotides.

The polynucleotides of the present invention can be DNA, or RNA, or chimeric mixtures, or derivatives or modified versions thereof, single stranded, or double stranded. The oligonucleotide can be modified with a base moiety, sugar moiety, or phosphate backbone to improve, for example, molecular stability, hybridization, and the like.
This oligonucleotide can be attached to other adducts such as peptides (eg, to target host cell receptors in vivo) or factors that facilitate transport across the cell membrane (eg, Letsinger et al., Proc. Natl. Acad).
. Sci. U. S. A. 86: 6553-6556 (1989); Lemait
re et al., Proc. Natl. Acad. Sci. 84: 648-652 (19
87); PCT publication number WO88 / 09810 (published December 15, 1988)
Or the blood brain barrier (eg, PCT Publication No. WO 89/101).
34 (published April 25, 1988)), hybridization-triggered cleavag.
e agent) (eg, Krol et al., BioTechniques, 6: 9
58-976 (1988)), or intercalating agents (see, for example, Zon, Pharm. Res. 5: 539-549 (1988)). For this purpose, the oligonucleotide is a separate molecule (eg,
Peptide, hybridization-induced crosslinking agent, transport agent, hybridization-induced cleavage agent, etc.).

The antisense oligonucleotide can comprise at least one modified base moiety, which base moiety is selected from the group including but not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2
-Thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylcuocin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D- Mannosyl cuocin, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2
-Methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v
), Wybutoxosine, pseudouracil, cuocin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxy Acetic acid (v), 5-methyl-2-thiouracil,
3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3)
w, and 2,6-diaminopurine.

Antisense oligonucleotides can also include at least one modified sugar moiety selected from the group including but not limited to: arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including but not limited to: phosphorothioate, phosphorodithioate, phosphoramidothioate , Phosphoramidates, phosphorodiamidates, methylphosphonates, alkylphosphotriesters, and formacetals or analogs thereof.

In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. a-anomeric oligonucleotides form specific double-stranded hybrids with complementary RNA, and their strands are parallel to each other as opposed to the normal b-unit (Gautier et al., Nucl. Acids Re
s. 15: 6625-6641 (1987)). This oligonucleotide is
2-0-methyl ribonucleotides (Inoue et al., Nucl. Acid
s Res. 15: 6131-6148 (1987)), or chimeric RNA
A DNA analogue (Inoue et al., FEBS Lett. 215: 327
-330 (1987)).

The polynucleotides of the invention can be prepared using standard methods known in the art (eg, automated D
NA synthesizer (such devices are Biosearch, Applied Bios
can be synthesized) by use of commercially available systems such as systems. For example,
Phosphorothioate oligonucleotides are prepared according to the method of Stein et al. (Nucl. A
cids Res. 16: 3209 (1988)), and methylphosphonate oligonucleotides can be synthesized using controlled pore glass (contro).
lled pore glass) polymer support (Sarin et al., Proc.
Natl. Acad. Sci. U. S. A. 85: 7448-7451 (19
88)) and the like.

On the other hand, antisense nucleotides complementary to the coding region sequences of the present invention can be used, with antisense nucleotides complementary to the transcribed untranslated region being most preferred.

Potential antagonists according to the present invention also include catalytic RNAs, ie ribozymes (eg PCT International Publication WO 90/11364, October 1990 4).
Published by Sarber et al., Science 247: 1222-1225 (19
90)). On the other hand, a ribozyme that cleaves mRNA with a site-specific recognition sequence can be used to destroy the mRNA corresponding to the polynucleotide of the present invention, but the use of a hammerhead ribozyme is preferred. Hammerhead ribozymes cleave mRNAs at positions determined by adjacent regions that form complementary base pairs with the target mRNA. The only requirement is that the target mRNA has the following two base sequences: 5'-UG-3 '. The construction and generation of hammerhead ribozymes is well known in the art, and Haseloff and Ge
rach, Nature, 334: 585-591 (1988). There are many potential hammerhead ribozyme cleavage sites within each nucleotide sequence disclosed in the sequence listing. Preferably, the ribozyme is such that the cleavage recognition site is located near the 5 ′ end of the mRNA corresponding to the polynucleotide of the invention; that is, increases efficiency and increases intracellular accumulation of non-functional mRNA transcripts. Manipulated to minimize.

In the case of an antisense approach, ribozymes of the invention can be composed of modified oligonucleotides (eg, improved for stability, targeting, etc.) and delivered in vivo to cells expressing the polynucleotides of the invention. It should be. A DNA construct encoding a ribozyme can be introduced into a cell in the same manner as described above for the introduction of an antisense encoding DNA. A preferred method of delivery “encodes” a ribozyme under the control of a strong constitutive promoter (eg, such as a pol III or pol II promoter).
Using a DNA construct so that the transfected cells destroy the endogenous message and produce an amount of ribozyme sufficient to inhibit translation. Since ribozymes are catalytic unlike antisense molecules, lower intracellular concentrations are required for efficiency.

Utilizing antagonist / agonist compounds, cell growth and proliferation of the polypeptides of the invention against neoplastic cells and tissues.
feration) effect may be inhibited. That stimulates tumor angiogenesis,
Thereby delaying or preventing abnormal cell growth and proliferation (eg in tumor formation or proliferation).

Antagonists / agonists can also be utilized to inhibit hypervascular disease and prevent proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of mitogenic activity of the polypeptides of the invention may also be required in cases such as restenosis after balloon angioplasty, for example.

Antagonists / agonists can also be utilized to prevent the growth of scar tissue during wound healing.

Antagonists / agonists may also be utilized to treat, prevent and / or diagnose the diseases described herein.

Accordingly, the present invention includes diseases, disorders and / or conditions (including but not limited to diseases, disorders and / or conditions enumerated throughout this application related to overexpression of a polynucleotide of the present invention). Is not treated), which is treated or prevented by administering to a patient (a) an antisense molecule against the polynucleotide of the invention and / or (b) a ribozyme against the polynucleotide of the invention. Provide a method. Invention and / or (b) Ribozymes relating to the polynucleotides of the invention.

(Other activities)
The polypeptides of the present invention revascularize ischemic tissue resulting from various disease states (eg, thrombosis, arteriosclerosis, and other cardiovascular conditions) as a result of the ability to stimulate vascular endothelial cell proliferation. It can be utilized in treatments for stimulation. These polypeptides can also be utilized to stimulate angiogenesis and limb remodeling as discussed above.

This polypeptide may also be used to treat wounds resulting from injury, burns, post-operative tissue repair, and ulcers. This is because they are mitogenic to various cells of different origin (eg, fibroblasts and skeletal muscle cells) and thus promote the repair or replacement of damaged or diseased tissue.

The polypeptides of the invention also stimulate neuronal growth and treat, prevent and prevent neuronal damage that occurs in certain neuronal disorders or neurodegenerative conditions (eg, Alzheimer's disease, Parkinson's disease, and AIDS-related complications). Can be used to diagnose. The polypeptides of the invention may have the ability to stimulate chondrocyte proliferation and thus enhance bone and periodontal remodeling and may be utilized for assistance in tissue or bone grafts .

The polypeptides of the invention can also be utilized to prevent skin aging due to sunburn by stimulating keratinocyte proliferation.

The polypeptides of the present invention can also be utilized to prevent hair loss. This is because members of the FGF family activate hair-forming cells and promote melanocyte proliferation. Along the same line, the polypeptides of the invention can be utilized to stimulate the growth and differentiation of hematopoietic and bone marrow cells when used in combination with other cytokines.

The polypeptides of the present invention can also be utilized to maintain organs prior to transplantation or can be used to support cell cultures of primitive tissues.

The polypeptides of the invention can also be utilized to induce tissue of mesodermal origin for differentiation in early embryos.

The polypeptides or polynucleotides and / or agonists or antagonists of the present invention may also increase or decrease embryonic stem cell differentiation or proliferation in addition to the hematopoietic lineage as discussed above.

The polypeptides or polynucleotides and / or agonists or antagonists of the present invention may also have mammalian characteristics (eg, height, weight, hair color,
It can be used to adjust eye color, skin, adipose tissue percentage, pigmentation, size, and shape (eg, cosmetic surgery). Similarly, the polypeptides or polynucleotides and / or agonists or antagonists of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and energy storage.

The polypeptides or polynucleotides and / or agonists or antagonists of the present invention may be biorhythmic, caricadic.
) Rhythm, depression (including depressive diseases, disorders and / or conditions), tendency to violence, pain tolerance, fertility (preferably by activin or inhibin-like activity), hormone or endocrine levels, appetite It can be used to alter the mental or physical state of a mammal by affecting sexual desire, memory, stress, or other cognitive quality.

The polypeptides or polynucleotides and / or agonists or antagonists of the present invention may also increase or decrease storage capacity, fat content, lipids, proteins, carbohydrates, vitamins, minerals, cofactors, or other nutritional components, for example. Can be used as a food additive or preservative.

(Other preferred embodiments)
Another preferred embodiment of the present invention comprises an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: X, wherein X is an arbitrary integer as defined in Table 1.

The sequence of consecutive nucleotides begins with a nucleotide at approximately the 5 ′ nucleotide position of the clone sequence, as defined for SEQ ID NO: X in Table 1,
Also preferred are nucleic acid molecules contained in the nucleotide sequence of SEQ ID NO: X, in a range of positions ending with nucleotides at approximately the 3 ′ nucleotide position of the clone sequence.

The sequence of consecutive nucleotides begins with a nucleotide at approximately 5 ′ nucleotide position of the start codon and ends with a nucleotide at approximately 3 ′ nucleotide position of the cloned sequence, as defined for SEQ ID NO: X in Table 1. Also preferred are nucleic acid molecules, which are included in the nucleotide sequence of SEQ ID NO: X within the range.

The sequence of consecutive nucleotides begins approximately at the 5 ′ nucleotide position of the first amino acid of the signal peptide, as defined for SEQ ID NO: X in Table 1, and approximately the 3 ′ nucleotide of the cloned sequence. Likewise preferred are nucleic acid molecules contained in the nucleotide sequence of SEQ ID NO: X, in the range of positions ending with the nucleotide at the position.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: X.

Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: X.

Further preferred embodiments are as defined for SEQ ID NO: X in Table 1,
Comprising a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: X beginning at about the nucleotide at the 5 ′ nucleotide position of the first amino acid of the signal peptide and ending at about the 3 ′ nucleotide position of the cloned sequence A nucleic acid molecule.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the complete nucleotide sequence of SEQ ID NO: X.

Also preferred is an isolated nucleic acid molecule that hybridizes to a nucleic acid molecule under stringent hybridization conditions, wherein the hybridizing nucleic acid molecule described above contains only A residues or T under stringent hybridization conditions. It does not hybridize to nucleic acid molecules having a nucleotide sequence consisting of only residues.

Also preferred is a composition of matter comprising a DNA molecule comprising a human cDNA clone identified by the cDNA clone ID in Table 1, which is contained in the material deposited with the American Type Culture Collection, and the cDNA described above The ATCC accession number shown in Table 1 is given for the clone ID.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the nucleotide sequence of a human cDNA clone identified by the cDNA clone ID in Table 1.
This DNA molecule is contained in the deposit given the ATCC deposit number shown in Table 1.

Also preferred is an isolated nucleic acid molecule wherein the sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence of the complete open reading frame sequence encoded by the human cDNA clone.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by the human cDNA clone.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 500 contiguous nucleotides in the nucleotide sequence encoded by the human cDNA clone.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the complete nucleotide sequence encoded by the human cDNA clone.

A further preferred embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of A nucleotide sequence of SEQ ID NO: X, wherein X is any integer as defined in Table 1; and identified by the cDNA clone ID in Table 1, and in Table 1 said cDNA clone A nucleotide sequence encoded by a human cDNA clone contained in a deposit having the ATCC accession number indicated for; said method comprising a sequence selected from the above group and at least one nucleic acid molecule in said sample; Comparing the nucleotide sequence with the nucleus in the sample; Sequence of molecules, comprising the step of determining whether at least 95% identical to the above selected sequence.

The method wherein the comparing the sequences comprises determining the degree of nucleic acid hybridization between a nucleic acid molecule in the sample and a nucleic acid molecule comprising the sequence selected from the group. Is also preferred. Similarly, the step of comparing the sequence comprises a nucleotide sequence determined from the nucleic acid molecule in the sample;
Also preferred is the above method, which is carried out by comparing the sequence selected from the above group. Nucleic acid molecules can include DNA molecules or RNA molecules.

A further preferred embodiment is a method for identifying a species, tissue, or cell type of a biological sample, said method comprising at least 50 contiguous nucleotides in a sequence selected from the group consisting of: Detecting a nucleic acid molecule (if any) in the sample comprising a nucleotide sequence that is at least 95% identical to the sequence: a nucleotide sequence of SEQ ID NO: X, wherein X is defined in Table 1 And A identified by the cDNA clone ID in Table 1 and indicated for the cDNA clone in Table 1
Nucleotide sequence encoded by a human cDNA clone contained in a deposit having a TCC accession number.

A method for identifying a species, tissue, or cell type of a biological sample can include detecting a nucleic acid molecule comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein At least one sequence is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the above group.

Also preferred is a method for diagnosing a pathological condition in a subject associated with an abnormal structure or expression of a gene encoding a secreted protein identified in Table 1, wherein the method is selected from the group consisting of: Detecting a nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in said sequence in a biological sample obtained from said subject (if any). The nucleotide sequence of SEQ ID NO: X, where X is any integer as defined in Table 1; and identified by the cDNA clone ID in Table 1 and indicated for the above cDNA clone in Table 1 Nucleotide encoded by human cDNA clone contained in deposit with ATCC deposit number Array.

A method for diagnosing a pathological condition can include detecting a nucleic acid molecule comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in the panel comprises the above Is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group.

Also preferred is a composition of matter comprising an isolated nucleic acid molecule, wherein the nucleotide sequence of the nucleic acid molecule comprises a panel of at least two nucleotide sequences, wherein at least one sequence in the panel comprises: At least 95% identical to a sequence of at least 50 consecutive nucleotides in a sequence selected from the group: nucleotide sequence of SEQ ID NO: X, wherein X is any integer as defined in Table 1; And a nucleotide sequence encoded by a human cDNA clone contained in the deposit identified by the cDNA clone ID in Table 1 and having the ATCC accession number indicated for the above cDNA clone in Table 1. Nucleic acid molecules can include DNA molecules or RNA molecules.

Also preferred is an isolated polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least about 10 contiguous amino acids in the amino acid sequence of SEQ ID NO: Y, wherein Y is defined in Table 1. Is any integer.

The sequence of consecutive amino acids begins at the residue of the first amino acid position of the secretory portion, as shown for SEQ ID NO: Y in Table 1, and at the residue of the last amino acid of the open reading frame. Sequence number Y in the range of the end position
Polypeptides included in the amino acid sequence of are also preferred.

Also preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of SEQ ID NO: Y.

Further preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of SEQ ID NO: Y.

Further preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to the complete amino acid sequence of SEQ ID NO: Y.

Identified by the cDNA clone ID in Table 1 and the above cDN in Table 1
Human c contained in the deposit with the ATCC deposit number indicated for the A clone
Further preferred is an isolated polypeptide comprising, in the complete amino acid sequence of the secreted protein encoded by the DNA clone, an amino acid sequence that is at least 90% identical to a sequence of at least about 10 consecutive amino acids.

Secretion encoded by the human cDNA clone contained in the deposit identified by the cDNA clone ID in Table 1 and having the ATCC accession number indicated for the cDNA clone in Table 1 above. Polypeptides included in the amino acid sequence of the secreted portion of the protein are also preferred.

Identified by the cDNA clone ID in Table 1 and the above c in Table 1
Included in the deposit with the ATCC accession number indicated for the DNA clone,
Also preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of the secreted portion of the protein encoded by the human cDNA clone.

Identified by the cDNA clone ID in Table 1 and the above c in Table 1
Included in the deposit with the ATCC accession number indicated for the DNA clone,
Also preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of the secreted portion of the protein encoded by the human cDNA clone.

Identified by the cDNA clone ID in Table 1 and the above c in Table 1
An isolated polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of the secreted portion of the protein encoded by the human cDNA clone contained in the deposit having the ATCC accession number indicated for the DNA clone is also preferable.

An isolated antibody that specifically binds to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: Further preferred: the amino acid sequence of SEQ ID NO: Y (where Y is any integer defined in Table 1); and the ATCC identified by the cDNA clone ID in Table 1 and indicated for this cDNA clone in Table 1 The complete amino acid sequence of the protein encoded by the human cDNA clone contained in the deposit with the accession number.

More preferred is a method for detecting in a biological sample a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: The amino acid sequence of number Y (where Y is any integer defined in Table 1); and having the ATCC accession number identified by the cDNA clone ID in Table 1 and indicated for this cDNA clone in Table 1 The complete amino acid sequence of the protein encoded by the human cDNA clone contained in the deposit; the method comprising comparing the amino acid sequence of at least one polypeptide molecule in the sample with a sequence selected from this group and The sequence of this polypeptide molecule in the sample is low At least ten pieces of the sequence of contiguous amino acids with 9
Determining whether they are 0% identical.

This step of comparing the amino acid sequence of at least one polypeptide molecule in the sample with a sequence selected from the group comprises at least 10 of the polypeptide in the sample in a sequence selected from the group consisting of: Also comprising the step of determining the degree of specific binding to an antibody that specifically binds to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of consecutive amino acids. Also preferred: the amino acid sequence of SEQ ID NO: Y, wherein Y
Is any integer as defined in Table 1); and the ATC identified by the cDNA clone ID in Table 1 and indicated for this cDNA clone in Table 1
The complete amino acid sequence of the protein encoded by the human cDNA clone contained in the deposit with the C deposit number.

Also preferred is the above method, wherein said step of comparing the sequence is performed by comparing the amino acid sequence determined from the polypeptide molecules in this sample with this sequence selected from this group.

Also preferred is a method of identifying the species, tissue or cell type of a biological sample, wherein the method comprises a polypeptide molecule in the sample (if the polypeptide is present, selected from the group consisting of: At least 10 in the sequence
Including an amino acid sequence that is at least 90% identical to the sequence of consecutive amino acids)
The amino acid sequence of SEQ ID NO: Y (where Y is any integer defined in Table 1); and the cDNA clone of Table 1 identified by the cDNA clone ID in Table 1 The complete amino acid sequence of the secreted protein encoded by the human cDNA clone contained in the deposit having the ATCC accession number indicated for.

Also preferred is the above-described method of identifying a biological sample species, tissue or cell type, wherein the method comprises detecting a polypeptide molecule comprising an amino acid sequence in a panel of at least two amino acid sequences. Wherein at least one sequence in the panel is at least one in a sequence selected from the above group.
It is at least 90% identical to the sequence of 0 consecutive amino acids.

Also preferred is a method of diagnosing a pathological condition in a subject that is associated with an abnormal structure or expression of a gene encoding a secreted protein identified in Table 1.
Wherein the method comprises detecting a polypeptide molecule comprising an amino acid sequence in a panel of at least two amino acid sequences in a biological sample obtained from the subject, wherein at least One sequence is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO: Y, where Y is defined in Table 1 Any integer); and cDNA clone I in Table 1
ATCC identified by D and shown for this cDNA clone in Table 1
The complete amino acid sequence of the secreted protein encoded by the human cDNA clone contained in the deposit with the accession number.

In any of these methods, detecting the polypeptide molecule comprises
Using an antibody.

A nucleotide sequence that is at least 95% identical to a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 consecutive amino acids in a sequence selected from the group consisting of: Also preferred is an isolated nucleic acid molecule comprising: the amino acid sequence of SEQ ID NO: Y, wherein
Y is any integer defined in Table 1); and the AT identified by the cDNA clone ID in Table 1 and indicated for this cDNA clone in Table 1
The complete amino acid sequence of the secreted protein encoded by the human cDNA clone contained in the deposit with the CC accession number.

Also preferred are isolated nucleic acid molecules in which the nucleotide sequence encoding the polypeptide is optimized for expression of the polypeptide in a prokaryotic host.

Also preferred is an isolated nucleic acid molecule wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: amino acid sequence of SEQ ID NO: Y, wherein Y is any integer defined in Table 1 And the complete amino acid sequence of the secreted protein encoded by the human cDNA clone contained in the deposit identified by the cDNA clone ID in Table 1 and having the ATCC accession number indicated for this cDNA clone in Table 1.

More preferred is a method for producing a recombinant vector comprising the step of inserting any of the isolated nucleic acid molecules described above into a vector. Also preferred is a recombinant vector produced by this method. Also preferred is a method of making a recombinant host cell comprising the step of introducing a vector into the host cell, as well as a recombinant host cell produced by this method.

Also preferred is a method of making an isolated polypeptide comprising culturing the recombinant host cell under conditions such that the polypeptide is expressed, and recovering the polypeptide. The recombinant host cell is a eukaryotic cell and the polypeptide produces an isolated polypeptide that is a secreted portion of a human secreted protein comprising an amino acid sequence selected from the group consisting of This method is also preferred: the amino acid sequence of SEQ ID NO: Y starting at the residue of the first amino acid position of the secretory part of SEQ ID NO: Y, where Y is an integer as described in Table 1 and SEQ ID NO: This position of the first amino acid of the secretory part of Y is defined in Table 1); and included in the deposit identified by the cDNA clone ID in Table 1 and having the ATCC accession number indicated for this cDNA clone in Table 1 The amino acid sequence of the secreted portion of the protein encoded by the human cDNA clone. Also preferred is an isolated polypeptide produced by this method.

Also preferred is a method of treating an individual in need of increased levels of secreted protein activity. Here, the method includes a pharmaceutical comprising an amount of an isolated polypeptide, polynucleotide, or antibody of the invention effective to increase the level of activity of the protein in the individual. Administering a pharmaceutical composition.

The applications cited above are used in a wide variety of hosts. Such hosts include humans, mice, rabbits, goats, guinea pigs, camels, horses,
Examples include, but are not limited to, mice, rats, hamsters, pigs, micro pigs, chickens, goats, cows, sheep, dogs, cats, non-human primates, and humans. In certain embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In the most preferred embodiment, the host is a human.

In certain embodiments of the invention, for each “Contig ID” listed in the fourth column of Table 7, preferably the nucleotide sequence referenced in the fifth column of Table 7, and the general formula ab Wherein one or more polynucleotides comprise or consist of the nucleotide sequence described by wherein a and b are uniquely determined for the corresponding SEQ ID NO: X referenced in column 3 of Table 7 Excluded. More specific embodiments are directed to polynucleotide sequences that exclude 1, 2, 3, 4, or more of the specific polynucleotide sequences referenced in the fifth column of Table 7. This table in no way is meant to include all of the sequences that can be excluded by the general formula, it is merely an illustrative example. All documents available through these registrations are hereby incorporated by reference in their entirety.

概して、本発明を記載してきたが、例示の目的のために提供され、そして限定
を意図しない、以下の実施例を参照することによって、本発明はより容易に理解
される。

Although the present invention has been generally described, the present invention will be more readily understood by reference to the following examples, which are provided for purposes of illustration and are not intended to be limiting.

(Example)
Example 1: Isolation of selected cDNA clones from deposited samples
Each cDNA clone in the cited ATCC deposit is contained in a plasmid vector. Table 1 identifies the vectors used to construct the cDNA library from which each clone was isolated. In many cases, the vector used to construct the library is a phage vector from which the plasmid has been excised. The table immediately below correlates the relevant plasmid for each phage vector used in constructing the cDNA library. For example, if a particular clone is identified in Table 1 as being isolated in the vector “Lambda Zap”, the corresponding deposited clone is “pBluescript”.

ベクターＬａｍｂｄａ Ｚａｐ（米国特許第５，１２８，２５６号および同第
５，２８６，６３６号）、Ｕｎｉ−Ｚａｐ ＸＲ（米国特許第５，１２８，２５
６号および同第５，２８６，６３６号）、Ｚａｐ Ｅｘｐｒｅｓｓ（米国特許第
５，１２８，２５６号および同第５，２８６，６３６号）、ｐＢｌｕｅｓｃｒｉ
ｐｔ（ｐＢＳ）（Ｓｈｏｒｔ，Ｊ．Ｍ．ら、Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅ
ｓ．１６：７５８３−７６００（１９８８）；Ａｌｔｉｎｇ−Ｍｅｅｓ，Ｍ．Ａ
．およびＳｈｏｒｔ，Ｊ．Ｍ．、Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． １７
：９４９４（１９８９））ならびにｐＢＫ（Ａｌｔｉｎｇ−Ｍｅｅｓ，Ｍ．Ａ．
ら、Ｓｔｒａｔｅｇｉｅｓ ５：５８−６１（１９９２））は、Ｓｔｒａｔａｇ
ｅｎｅ Ｃｌｏｎｉｎｇ Ｓｙｓｔｅｍｓ，Ｉｎｃ．、１１０１１ Ｎ．Ｔｏｒ
ｒｅｙ Ｐｉｎｅｓ Ｒｏａｄ、Ｌａ Ｊｏｌｌａ，ＣＡ，９２０３７から市販
されている。ｐＢＳは、アンピシリン耐性遺伝子を含み、そしてｐＢＫはネオマ
イシン耐性遺伝子を含む。両方とも、Ｅ．ｃｏｌｉ株ＸＬ−１ Ｂｌｕｅ（これ
もまた、Ｓｔｒａｔａｇｅｎｅから入手可能である）に形質転換され得る。ｐＢ
Ｓは、ＳＫ＋、ＳＫ−、ＫＳ＋およびＫＳの４形態で入荷する。ＳおよびＫとは
、ポリリンカー領域（「Ｓ」とはＳａｃＩであり、そして「Ｋ」とは、ＫｐｎＩ
のことであり、これらは、リンカーのそれぞれの各末端での最初の部位である）
に隣接するＴ７およびＴ３プライマー配列に対するポリリンカーの配向をいう。
「＋」または「−」とは 、ある方向においてｆ１ ｏｒｉから開始される一本
鎖レスキューがセンス鎖ＤＮＡを生成し、そして他方においてアンチセンスであ
るようなｆ１複製起点（「ｏｒｉ」）の配向をいう。

Vector Lambda Zap (US Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (US Pat. No. 5,128,25)
6 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript.
pt (pBS) (Short, JM et al., Nucleic Acids Re
s. 16: 7583-7600 (1988); Alting-Mees, M .; A
. And Short, J .; M.M. Nucleic Acids Res. 17
: 9494 (1989)) and pBK (Alting-Mees, MA.
Et al, Strategies 5: 58-61 (1992)).
ene Cloning Systems, Inc. 11011 N.R. Tor
commercially available from rey Pines Road, La Jolla, CA, 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Both can be found in E.C. can be transformed into E. coli strain XL-1 Blue (also available from Stratagene). pB
S arrives in four forms: SK +, SK−, KS + and KS. S and K are polylinker regions (“S” is SacI and “K” is KpnI
And these are the first sites at each end of each of the linkers)
Refers to the orientation of the polylinker relative to the T7 and T3 primer sequences adjacent to.
“+” Or “−” refers to the orientation of the f1 origin of replication (“ori”) such that a single-stranded rescue starting from f1 ori in one direction produces sense strand DNA and the other is antisense. Say.

Vectors pSport1, pCMVSPort 2.0 and pCMVSpo
rt 3.0 from Life Technologies, Inc. , P.M. O. Box
6009, from Gaithersburg, MD 20897. All Sport vectors contain an ampicillin resistance gene and coli strain D
H10B, which is also available from Life Technologies, can be transformed. (For example, Gruber, CE et al., Focus.
15:59 (1993)). Vector lafmid BA (Ben
to Soares, Columbia University, NY) contains an ampicillin resistance gene and E. coli. E. coli strain XL-1 Blue can be transformed. Vector pCR® 2.1 (this is Invitroge
n, 1600 Faraday Avenue, Carlsbad, CA 92
Contains the ampicillin resistance gene, and E. c
oli strain DH10B (available from Life Technologies) can be transformed (eg, Clark, JM, Nuc. Acids).
Res. 16: 9677-9686 (1988) and Mead, D .; Bi
o / Technology 9: (See 1991). Preferably, the polynucleotides of the invention do not include the phage vector sequences identified for the particular clone in Table 1, as well as the corresponding plasmid vector sequences shown above.

The deposited material in the sample given the ATCC accession number for any given cDNA clone cited in Table 1 also contains one or more additional plasmids (each of which is a different cDNA clone from that given clone). including)
Can be included. Thus, deposits that share the same ATCC accession number include at least a plasmid for each cDNA clone identified in Table 1. Typically, a sample of each ATCC deposit cited in Table 1 contains a mixture of approximately equal amounts (by weight) of about 50 plasmid DNAs, each containing a different cDNA clone; , Such deposit samples are more or less than 50 c
Plasmids for DNA clones (up to about 500 cDNA clones) can be included.

Two approaches can be used to isolate the clone from the deposited sample of plasmid DNA cited for the particular clone in Table 1. First, the plasmid is isolated directly by screening clones using a polynucleotide probe corresponding to SEQ ID NO: X.

In particular, specific polynucleotides having 30-40 nucleotides are synthesized using an Applied Biosystems DNA synthesizer according to the reported sequence. Oligonucleotides, for example, with ³² P-γ-ATP, T
Label with 4 polynucleotide kinase and purify according to conventional methods. (For example, Maniatis et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Harb
or Press, Cold Spring, NY (1982)). Plasmid mixtures can be prepared using techniques known to those skilled in the art (eg, techniques provided by vector suppliers or related publications or patents cited above).
To a suitable host as described above (eg, XL-1 Blue (Strat
agene)). Transformants are plated on 1.5% agar plates (containing an appropriate selection agent such as ampicillin) at a density of about 150 transformants (colony) per plate. These plates can be prepared using conventional methods for bacterial colony screening (eg, Sambrook et al., M
molecular Cloning: A Laboratory Manual
, 2nd edition, (1989), Cold Spring Harbor Labor.
screen) using nylon membranes according to art press (1.93 to 1.104) or other techniques known to those skilled in the art.

Alternatively, both ends of SEQ ID NO: X (ie, 5′N of the clone as defined in Table 1)
17-2 derived from within the region of SEQ ID NO: X surrounded by T and 3′NT
Two primers of 0 nucleotides are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is performed under conventional conditions, for example, in 25 μl of a reaction mixture with 0.5 μg of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl ₂ , 0.01% (w / v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 units of Taq polymerase. . 35 cycle PC
R (denaturation at 94 ° C. for 1 minute; annealing at 55 ° C. for 1 minute; extension at 72 ° C. for 1 minute) is performed using a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis, and the expected molecular weight DNA band is excised and purified. PCR product
Confirm the selected sequence by subcloning and sequencing the NA product.

Several methods are available for the identification of 5 ′ non-coding or 3 ′ non-coding portions of genes that may not be present in the deposited clone. These methods include, but are not limited to: filter probe search, clone enrichment using specific probes, and 5 ′ and 3 well known in the art.
'A protocol similar or identical to the' RACE 'protocol. For example, 5 '
A method similar to RACE can be used to generate a 5 ′ end lacking the desired full-length transcript (Frommont-Racine et al., Nucleic A
cids Res. 21 (7): 1683-1684 (1993)).

Briefly, a particular RNA oligonucleotide is ligated to the 5 ′ end of a population of RNA that probably contains a full-length gene RNA transcript. PCR the 5 'portion of the desired full-length gene using a primer set that includes a primer specific for the ligated RNA oligonucleotide and a primer specific for the known sequence of the gene of interest.
Amplify. The amplified product can then be sequenced and used to generate a full-length gene.

This above method starts with total RNA isolated from the desired source, but poly A + RNA can also be used. The RNA preparation can then be treated with phosphatase if necessary to degrade or damage RN that can interfere with subsequent RNA ligase steps.
The 5 'phosphate group of A can be eliminated. The phosphatase should then be inactivated and the RNA should be treated with tobacco acid pyrophosphatase to remove the cap structure present at the 5 ′ end of the messenger RNA.
This reaction leaves a 5 'phosphate group at the 5' end of the cleaved RNA that can then be ligated to the RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strand cDNA synthesis using gene-specific oligonucleotides. First strand synthesis reaction product
Use as a template for PCR amplification of the desired 5 'end using primers specific for the ligated RNA oligonucleotide and primers specific for the known sequence of the gene of interest. The resulting product is then sequenced and analyzed to confirm that the 5 ′ end sequence belongs to the desired gene.

(Example 2: Isolation of genomic clone corresponding to polynucleotide)
Human Genome P1 Library (Genomic Systems, Inc.)
Are screened by PCR using primers selected for the cDNA sequence corresponding to SEQ ID NO: X according to the method described in Example 1 (Sa
See also mbrook).

(Example 3: Tissue distribution of polypeptide)
The tissue distribution of mRNA expression of the polynucleotides of the present invention, inter alia, Samb
Determined using the protocol for Northern blot analysis described by look et al. For example, cDNA produced by the method described in Example 1
The probe is attached to the rediprime ^™ DNA labeling system.
(Amersham Life Science) using, according to the manufacturer's instructions, labeled with P ^32. After labeling, the probe was connected to CHROMA SPIN-100
Purify using ^TM column (Clontech Laboratories, Inc.) according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to test various human tissues for mRNA expression.

Multiple tissue northern (MTN) blots (Clontech) containing various human tissues (H) or human immune system tissues (IM) were prepared using the manufacturer's protocol number P using ExpressHyb ^™ hybridization solution (Clontech).
Test with labeled probe according to T1190-1. After hybridization and washing, the blot is mounted and exposed to film overnight at -70 ° C and the film is developed according to standard procedures.

(Example 4: Chromosome mapping of polynucleotide)
A set of oligonucleotide primers is designed according to the sequence at the 5 ′ end of SEQ ID NO: X. This primer preferably spans about 100 nucleotides.
This primer set is then used for polymerase chain reaction under the following set of conditions: 95 ° C. for 30 seconds; 56 ° C. for 1 minute; 70 ° C. for 1 minute. This cycle is repeated 32 times, followed by one cycle at 70 ° C. for 5 minutes. A somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Inc)
In addition, human, mouse, and hamster DNA are used as templates. The reaction is analyzed on either an 8% polyacrylamide gel or a 3.5% agarose gel. Chromosome mapping is approximately 100 in a specific somatic cell hybrid.
Determined by the presence of the bp PCR fragment.

(Example 5: Bacterial expression of polypeptide)
A polynucleotide encoding a polypeptide of the invention is amplified using PCR oligonucleotide primers corresponding to the 5 ′ and 3 ′ ends of the DNA sequence, as outlined in Example 1, to synthesize an insert fragment. The primer used to amplify the cDNA insert should preferably contain restriction sites such as BamHI and XbaI at the 5 ′ end of the primer in order to clone the amplification product into the expression vector. For example, BamHI and XbaI are expressed as bacterial expression vectors pQE-9 (Qiagen, Inc., Chatsworth,
Corresponds to the restriction enzyme site of CA). This plasmid vector is resistant to antibiotics (
Amp ^r ), bacterial origin of replication (ori), IPTG regulatable promoter /
Encodes operator (P / O), ribosome binding site (RBS), 6-histidine tag (6-His), and restriction enzyme cloning site.

The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated to the pQE-9 vector while maintaining the reading frame initiated in bacterial RBS. The ligation mixture is then transformed into plasmid pR that expresses the lacI repressor and confers resistance to kanamycin (Kan ^r ).
E. including multiple copies of EP4. coli strain M15 / rep4 (Qiagen,
Inc. ) Is used for transformation. Transformants are identified by their ability to grow on LB plates and ampicillin / kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.

Clones containing the desired construct were identified as Amp (100 μg / ml) and Kan (
Overnight in liquid culture in LB medium supplemented with 25 μg / ml) (O / N)
Proliferate. An O / N culture is used to inoculate a large culture at a ratio of 1: 100 to 1: 250. Cells are treated with an optical density 600 (OD) between 0.4 and 0.6.
. ⁶⁰⁰ ). IPTG (isopropyl-BD-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG is lacI
Induced by repressor inactivation, removes P / O obstacles and leads to increased gene expression.

Cells are grown for an additional 3-4 hours. The cells are then centrifuged (6000 ×
g for 20 minutes). Cell pellets are chaotropic agents 6
Dissolve in M guanidine HCl by stirring at 4 ° C. for 3-4 hours.
Cell debris is removed by centrifugation and the supernatant containing the polypeptide is removed from a nickel-nitrilotriacetic acid (“Ni-NTA”) affinity resin column (QIA).
GEN, Inc. Load from above. Proteins with a 6 × His tag bind with high affinity to Ni-NTA resin and can be purified in a simple one-step procedure (see The QIA expressionist (19
95) QIAGEN, Inc. , See above).

Briefly, the supernatant was loaded onto a 6M guanidine-HCl, pH 8 column and the column was first washed with 10 volumes of 6M guanidine-HCl, pH 8,
It is then washed with 10 volumes of 6M guanidine-HCl, pH 6, and finally the polypeptide is eluted with 6M guanidine-HCl, pH 5.

The purified protein is then added to phosphate buffered saline (PBS) or 5
Regenerate by dialyzing against 0 mM sodium acetate, pH 6 buffer and 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on a Ni-NTA column. The recommended conditions are as follows: regeneration using a linear gradient of 6M-1M urea in 500 mM NaCl, 20% glycerol, 20 mM Tris / HCl pH 7.4 with protease inhibitors. Regeneration should take over 1.5 hours. After playback,
Protein is eluted by addition of 250 mM imidazole. Imidazole, PBS or 50 mM sodium acetate pH 6 buffer and 200 mM
Removed by final dialysis step against NaCl. Purified protein
Store at 0 ° C. or freeze at −80 ° C.

In addition to the expression vectors described above, the present invention further includes an expression vector called pHE4a, comprising a phage operator and promoter element operably linked to the polynucleotide of the present invention (ATCC Accession No. 209645).
, Deposited on February 25, 1998). This vector includes: 1) the neomycin phosphotransferase gene as a selectable marker, 2) E. coli. E. coli origin of replication, 3) T5 phage promoter sequence, 4) two lac operator sequences, 5) Shine-Dalgarno sequence, and 6) lactose operon repressor gene (laqIq). The origin of replication (oriC) is pUC19 (LTI, Ga
from itersburg, MD). Promoter and operator sequences are made synthetically.

Restrict the vector with NdeI and XbaI, BamHI, XhoI, or Asp718, run the restriction product on a gel, and remove the larger fragment (the stuffer fragment should be about 310 base pairs) By isolation, the DNA can be inserted into pHEa. D
The NA insert was ligated with NdeI (5 ′ primer) and XbaI, BamHI, Xh
PCR with restriction sites for oI or Asp718 (3 ′ primer)
Generated according to the PCR protocol described in Example 1 using primers.
The PCR insert is gel purified and restricted with a compatible enzyme. The insert and vector are ligated according to standard protocols.

Engineered vectors can be easily replaced in the above protocol to express proteins in bacterial systems.

(Example 6: Purification of polypeptide from inclusion bodies)
Another method below is that E. coli, when the polypeptide is present in the form of inclusion bodies. co
It can be used to purify polypeptides expressed in li. Unless otherwise specified, all the following steps are performed at 4-10 ° C.

E. After completion of the production phase of E. coli fermentation, the cell culture is cooled to 4-10 ° C. and the cells are harvested by continuous centrifugation at 15,000 rpm (Heraeus Septech). Based on the expected yield of protein per unit weight of cell paste and the amount of purified protein required, the appropriate amount of cell paste (by weight) is adjusted to 100 mM Tris, 50 mM EDTA, pH 7.
Suspend in buffer solution containing 4. Cells are dispersed into a homogeneous suspension using a high shear mixer.

The cells are then microfluidized (microfluidizer (
Microfluidics, Corp. Or APV Gaulin, Inc
. ) Twice through the solution at 4000-6000 psi.
The homogenate is then mixed with a NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000 × g for 15 minutes. The resulting pellet was added to 0.5 M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.
Wash again using 4.

The washed inclusions obtained were washed with 1.5 M guanidine hydrochloride (GuHCl) for 2 to
Solubilize for 4 hours. After centrifugation at 7000 × g for 15 minutes, the pellet is discarded and the supernatant containing the polypeptide is incubated overnight at 4 ° C. for further Gu
Allows HCl extraction.

Following high speed centrifugation (30,000 × g) to remove insoluble particles, Gu
HCl solubilized protein was extracted with GuHCl extract, 50 mM sodium, pH
Refold by rapidly mixing with 20 volumes of buffer containing 4.5, 150 mM NaCl, 2 mM EDTA with vigorous stirring. The refolded diluted protein solution is kept at 4 ° C. without mixing for 12 hours prior to further purification steps.

To clarify the refolded polypeptide solution, a pre-prepared tangential filtration unit equipped with a 0.16 μm membrane filter with appropriate surface area equilibrated with 40 mM sodium acetate, pH 6.0 (eg, Filtro
n). The filtered sample is added to a cation exchange resin (eg, Poros
(HS-50, Perseptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0, and 250 mM, 500 mM, 1000 mM, and 1500 mM NaC in the same buffer.
Elute in a stepwise fashion. The absorbance at 280 nm of the eluate is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.

The fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample was then added to a previously prepared strong anion (Poros HQ
-50, Perseptive Biosystems) exchange resin and weak anion (Poros CM-20, Perseptive Biosystems)
) Load into a set of serial columns of exchange resin. The column is equilibrated with 40 mM sodium acetate, pH 6.0. Both columns were loaded with 40 mM sodium acetate,
Wash with pH 6.0, 200 mM NaCl. The CM-20 column is then replaced with 10
Linear gradient of column volume (0.2 M NaCl, 50 mM sodium acetate, pH
Elution with 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5). Fractions are collected under constant A ₂₈₀ monitoring of the eluate. The fractions containing the polypeptide (eg, found by 16% SDS-PAGE) are then pooled.

The resulting polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. When 5 μg of purified protein is loaded, no major mixed band should be observed from a Coomassie blue stained 16% SDS-PAGE gel. The purified protein is also endotoxin / LPS
Can be tested for contamination and typically the LPS content is less than 0.1 ng / ml according to the LAL assay.

Example 7 Cloning and Expression of Polypeptide in Baculovirus Expression System
In this example, the plasmid shuttle vector pA2 is used to insert a polynucleotide into a baculovirus and express the polypeptide. This expression vector is an Autographa californica nuclear polynuclear virus (
AcMNPV) strong polyhedrin promoter, followed by BamHI, Xba
Convenient restriction sites such as I and Asp718 are included. Simian virus 40
The polyadenylation site of (“SV40”) is used for efficient polyadenylation. For easy selection of recombinant viruses, this plasmid was transformed into E. coli under the control of a weak Drosophila promoter in the same orientation. a β-galactosidase gene from E. coli followed by a polyadenylation signal for the polyhedrin gene. The inserted gene is flanked on both sides by the viral sequence for cell-mediated homologous recombination with wild-type viral DNA, producing a viable virus that expresses the cloned polynucleotide.

Many other baculovirus vectors (eg, pAc373, pVL941)
, And pAcIM1), as will be readily understood by those skilled in the art, signals in which the construct is properly positioned for transcription, translation, secretion, etc. (including signal peptides and in-frame AUG, if required) Can be used instead of the vectors described above. Such vectors include, for example, Lucko
w et al., Virology 170: 31-39 (1989).

Specifically, the cDNA sequence contained in the deposited clone containing the AUG start codon and naturally bound to the leader sequence identified in Table 1 was amplified using the PCR protocol described in Example 1. Let If a naturally occurring signal sequence is used to produce a secreted protein, the pA2 vector does not require a second signal peptide. Alternatively, Summers et al., “A Manual o
f Methods for Baculovirus Vectors an
d Insect Cell Culture Procedures ", Te
xas Agricultural Experimental Statio
n Bulletin NO. : 1555 (1987) can be used to modify this vector to include a baculovirus leader sequence (pA2GP).

Amplified fragments are obtained from commercial kits (“Geneclean”, BIO
101 Inc. La Jolla, Ca. To isolate from a 1% agarose gel. This fragment is then digested with the appropriate restriction enzymes and purified again on a 1% agarose gel.

This plasmid can be digested with the corresponding restriction enzymes and, if necessary, dephosphorylated with calf intestinal phosphatase using conventional procedures known in the art. This DNA is then converted into a commercially available kit (“Geneclean” BIO 10
1 Inc. La Jolla, Ca. To isolate from a 1% agarose gel.

This fragment and the dephosphorylated plasmid are ligated together using T4 DNA ligase. E. E. coli HB101 cells or XL-1 Blu
e (Stratagene Cloning Systems, La Joll
a, CA) other suitable E. cells such as cells. E. coli hosts are transformed with the ligation mixture and spread on culture plates. Bacteria containing this plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.

5 μg of plasmid containing this polynucleotide was added to Felgner et al., Pr.
oc. Natl. Acad. Sci. USA 84: 7413-7417 (19
87) using 1.0 μg of commercially available linearized baculovirus DNA (“BaculoGold ^™ baculovi”).
rus DNA ", Pharmingen, San Diego, CA). 1 μg of BaculoGold ^™ viral DNA and 5 μg of plasmid were mixed with 50 μl of serum-free Grace's medium (Life Tech
nologies Inc. , Gaithersburg, MD) in a sterile well of a microtiter plate. Thereafter, 10 μl Lipofectin and 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. The transfection mixture was then transferred to Sf9 insect cells (A) seeded on 35 mm tissue culture plates supplemented with 1 ml of serum-free Grace's medium.
TCC CRL 1711). The plate is then incubated for 5 hours at 27 ° C. The transfection solution is then removed from the plate and 1 ml of Grace insect medium supplemented with 10% fetal calf serum is added. The culture is then continued for 4 days at 27 ° C.

After 4 days the supernatant is collected and a plaque assay is performed as described by Summers and Smith (supra). “Blue Gal” (Life
Technologies Inc. , Gaithersburg) allows easy identification and isolation of gal-expressing clones (resulting in blue-stained plaques) (details of this type of “plaque assay”) The description is also available from Life Technologies Inc.,
Can be found in the user guide for insect cell culture and baculovirology distributed by Gaithersburg, pages 9-10). After appropriate incubation, the blue stained plaques are picked up with a micropipette tip (eg Eppendorf). The agar containing the recombinant virus is then
Resuspend in a microcentrifuge tube containing 00 μl Grace's medium and use a suspension containing recombinant baculovirus to infect Sf9 cells seeded in 35 mm dishes. After 4 days, the supernatants of these culture dishes are collected and then stored at 4 ° C.

To confirm polypeptide expression, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. These cells are treated with an infection efficiency of about 2 ("MO
I ") with a recombinant baculovirus containing this polynucleotide.
If radiolabeled protein is desired, the medium is removed after 6 hours and SF900 II medium without methionine and cysteine (Life T
technologies Inc. , Rockville, MD). 42 hours later, 5 μCi of ³⁵ S-methionine and 5 μCi
³⁵ S-cysteine (available from Amersham) is added. Cells are further incubated for 16 hours and then harvested by centrifugation. Proteins in the supernatant as well as intracellular proteins are analyzed by SDS-PAGE and then by autoradiography (when radiolabeled).

The amino terminal sequence of the produced protein can be determined using micro sequencing of the amino terminal amino acid sequence of the purified protein.

(Example 8: Expression of polypeptide in mammalian cells)
The polypeptides of the present invention can be expressed in mammalian cells. A typical mammalian expression vector contains a promoter element that mediates the initiation of transcription of mRNA, a protein coding sequence, and signals necessary for transcription termination and transcript polyadenylation. Further elements are the enhancer Kozak (Koz
ak) contains the sequence and intervening sequences adjacent to the donor and acceptor sites for RNA splicing. Very efficient transcription is achieved by early and late promoters from SV40, retroviruses (eg RSV, HTLVI, H
IVI) long terminal repeat (LTR), and cytomegalovirus (CMV)
Of the early promoter. However, intracellular elements (eg
A human actin promoter) can also be used.

Suitable expression vectors for use in practicing the present invention include, for example, pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVc.
at (ATCC 37152), pSV2dhfr (ATCC 37146),
Vectors such as pBC12MI (ATCC 67109), pCMVSPort2.0, and pCMVSPort3.0 are included. Mammalian host cells that can be used include human Hela cells, 293 cells, H9 cells and Jurka.
t cells, mouse NIH3T3 cells and C127 cells, Cos 1 cells, Co
s7 cells and CV1 cells, quail QC1-3 cells, mouse L cells, and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide comprises a polynucleotide integrated into the chromosome,
It can be expressed in stable cell lines. Co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is
Useful for the development of cell lines with hundreds or even thousands of copies of the gene of interest (eg, Alt, FW et al., J. Biol. Chem. 253: 1357).
-1370 (1978); Hamlin, J. et al. L. And Ma, C.I. , Bioc
hem. et Biophys. Acta, 1097: 107-143 (199
0); Page, M .; J. et al. And Sydenham, M .; A. Et al, Biotec
hnology, 9: 64-68 (1991)). Another useful selectable marker is the enzyme glutamine synthase (GS) (Murphy et al. Bi).
ochem J.H. 227: 277-279 (1991); Bebbington
Et al., Bio / Technology, 10: 169-175 (1992)). Using these markers, mammalian cells are grown in selective media and the cells with the highest resistance are selected. These cell lines contain the amplified gene integrated into the chromosome. Chinese hamster ovary (CHO) and NS
O cells are often used for protein production.

Expression vectors pC4 (ATCC accession number 209646) and pC6 (AT), which are derivatives of plasmid pSV2-dhfr (ATCC accession number 37146)
CC accession number 209647) is a rous sarcoma virus (Cullen et al., Mol.
economic and Cellular Biology, 438-447 (
March 1985)) strong promoter (LTR), and a fragment of the CMV-enhancer (Boshart et al., Cell 41: 521-530 (1985)). For example, multiple cloning sites with BamHI, XbaI, and Asp718 restriction sites facilitate the cloning of the gene of interest. This vector also contains 3 ′ introns, polyadenylation and termination signals of the rat preproinsulin gene, and S
Contains the mouse DHFR gene under the control of the V40 early promoter.

Specifically, for example, plasmid pC6 is digested with an appropriate restriction enzyme,
It is then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The vector is then isolated from a 1% agarose gel.

The polynucleotides of the invention are amplified according to the protocol outlined in Example 1. If a naturally occurring signal sequence is used for the production of a secreted protein, the vector need not contain a second signal peptide. Or
If no naturally occurring signal sequence is used, the vector can be modified to include a heterologous signal sequence (see, eg, WO 96/34891).

Amplified fragments were obtained from commercial kits (“Geneclean”, BIO 10
1 Inc. La Jolla, Ca. To isolate from a 1% agarose gel. This fragment is then digested with the appropriate restriction enzymes and purified again on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzymes and purified on a 1% agarose gel. The isolated fragment and dephosphorylated vector are then ligated with T4 DNA ligase. Then E. E. coli HB101 or XL-1 Blue cells are transformed and bacteria containing the fragment inserted into plasmid pC6 are identified using, for example, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used for transfection. 5 μg of expression plasmid pC6 a pC4 was added to 0.5 μg of plasmid p using lipofectin (Felner et al., Supra).
Co-transfect with SVneo. Plasmid pSV2-neo contains the Tn5-derived neo gene which encodes an enzyme conferring resistance to a group of antibiotics including G418, which is a dominant selectable marker. Cells are seeded in alpha minus MEM supplemented with 1 mg / ml G418. Two days later, the cells were trypsinized and 10, 25, or 50 ng / ml methotrexate and 1 m
Seed in hybridoma cloning plates (Greiner, Germany) in α minus MEM supplemented with g / ml G418. After about 10-14 days, a single clone was trypsinized and then using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM)
Inoculate in a 6-well petri dish or 10 ml flask. Then clones growing at the highest concentration of methotrexate were allowed to grow at higher concentrations (1 μM, 2 μM, 5
Transfer to a new 6-well plate containing methotrexate (μM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained that grow at a concentration of 100-200 μM. Expression of the desired gene product is analyzed, for example, by SDS-PAGE and Western blot, or by reverse phase HPLC analysis.

(Example 9: Protein fusion)
The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins can be used for various applications. For example, the fusion of a polypeptide of the invention to His tag, HA tag, protein A, IgG domain, and maltose binding protein facilitates purification (see Example 5; EP
See also A 394,827; Traunecker et al., Nature.
e, 331: 84-86 (1988)). Similarly, fusion to IgG-1, IgG-3, and albumin increases the half-life in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target proteins to specific intracellular localizations. On the other hand, covalent heterodimers or homodimers can increase or decrease the activity of the fusion protein. Fusion proteins can also create chimeric molecules with more than one function. Finally, the fusion protein can increase the solubility and / or stability of the fusion protein compared to a non-fusion protein. All types of fusion proteins described above can be made by modifying the following protocol that outlines the fusion of a polypeptide to an IgG molecule, or the protocol described in Example 5.

Briefly, the human Fc portion of an IgG molecule can be PCR amplified using primers that span the 5 ′ and 3 ′ ends of the sequences described below. These primers should also have convenient restriction enzyme sites that facilitate cloning into an expression vector (preferably a mammalian expression vector).

For example, when pC4 (Accession No. 209646) is used, the human Fc portion can be linked to a BamHI cloning site. Note that the 3 ′ BamHI site should be destroyed. Next, the vector containing the human Fc portion is B
The polynucleotide of the present invention, which was again restricted using amHI, linearized the vector, and isolated by the PCR protocol described in Example 1, was
Ligated to the amHI site. Note that the polynucleotide is cloned without a stop codon, otherwise no fusion protein is produced.

If a naturally occurring signal sequence is used to produce a secreted protein, pC4 does not require a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence (see, eg, WO 96/34891).
Human IgG Fc region:
GGGATCCGGAGCCCAAATCTTCTGACAAAAACTCACA
CATGCCCACCGTGCCCAGCACCCTGAATTCGAGGGTG
CACCGTCAGTTCTCTCCTTCCCCCAAAAACCCAAGG
ACACCCTCATGATCTCCCGGACTACTCTGAGGTCACAT
GCGTGGGTGTGGACGTAAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACCGTGGACGCGTGGGAGGTGC
ATAATGCCCAAGACAAAGCCCCGGGGAGGAGCAGTACA
ACAGCACGTACCCGTGTGGTCAGCGTCCTCACCGTCC
TGCACCAGGACTGGCTGAATGGCAAGGAGTACAGGT
GCAAGGTCCTCAACAAAGCCCCTCCCAACCCCCATCG
AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCCAGAG
AACCACAGGTGTACACCCTGCCCCCCATCCGGGATG
AGCTGACCAAGAACCAGGTCAGCTCGACCTGCCTG
TCAAAGGCTTCTATTCCAAGCGACATCGCCGTGGAGT
GGGAGAGCAATGGGGCAGCCGGAGAACAAACTACAGA
CCACGCCCTCCCGTGCTGGAACTCCGACGGCTCTCTTCT
TCCTCTACAGCAAGCTCACCGTGGGACAAGAGCAGGT
GGCAGCAGGGGAACGTTCTTCTCATGCTCCCGTGATGC
ATGAGGCTCTGCACAACCACTACACCAGCAGAGAGCC
TCTCCCTGTCCTCGGGTAAATGAGGTCGACGGCCCGC
GACTCTAGAGGAT (SEQ ID NO: 1)
(Example 10: Production of antibody from polypeptide)
The antibodies of the present invention can be prepared by various methods. (Current Pr
autocols, see Chapter 2). As one example of such a method, cells expressing a polypeptide of the invention are administered to an animal to induce production of serum containing polyclonal antibodies. In a preferred method, a secreted protein preparation is prepared and purified so that it is substantially free of natural contaminants. Such preparations are then introduced into animals in order to produce higher specific activity polyclonal antisera.

In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or protein binding fragments thereof). Such monoclonal antibodies are prepared using hybridoma technology (Kohler et al., Nature 25
6: 495 (1975); Kohler et al., Eur. J. et al. Immunol. 6:
511 (1976); Kohler et al., Eur. J. et al. Immunol. 6:29
2 (1976); Hammerling et al .: Monoclonal Antib.
odies and T-Cell Hybridomas, Elsevier
, N.M. Y. 563-681 (1981)). In general, such procedures involve immunizing an animal (preferably a mouse) with a polypeptide, or more preferably with a secretory polypeptide-expressing cell. Such cells can be cultured in any suitable tissue culture medium; however, preferably supplemented with 10% fetal calf serum (inactivated at about 56 ° C.) and about 10 g / l of nonessential amino acids, About 1,
Incubate in Earle's modified Eagle's medium supplemented with 000 U / ml penicillin and about 100 μg / ml streptomycin.

Such mouse splenocytes are extracted and fused with an appropriate myeloma cell line. Any suitable myeloma cell line can be used in accordance with the present invention;
It is preferred to use the parent myeloma cell line (SP2O) available from CC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and then Wands et al. (Gastroenterology 80: 225-232 (
1981)) and cloned by limiting dilution. The hybridoma cells obtained through such selection are then assayed to identify clones that secrete antibodies capable of binding to the polypeptide.

Alternatively, additional antibodies that can bind to the polypeptide can be generated in a two-step procedure using anti-idiotype antibodies. Such a method takes advantage of the fact that the antibody itself is an antigen and therefore it is possible to obtain an antibody that binds to a second antibody. According to this method, an animal (preferably a mouse) is immunized using a protein specific antibody. The splenocytes of such animals are then used to produce hybridoma cells. The hybridoma cells are then screened to identify clones that produce an antibody whose ability to bind to a protein-specific antibody can be blocked by the polypeptide. Such antibodies include anti-idiotypic antibodies against protein specific antibodies and are used to immunize animals to induce the formation of additional protein specific antibodies.

It will be apparent that Fab and F (ab ') 2 and other fragments of the antibodies of the present invention may be used in accordance with the methods described herein. Such fragments are typically produced by proteolytic cleavage using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab ′) 2 fragments). Is done. Alternatively, secreted protein-binding fragments can be produced by the application of recombinant DNA techniques or synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use “humanized” chimeric monoclonal antibodies. Such antibodies can be produced by using genetic constructs derived from hybridoma cells that produce the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. (For a review, see Morrison, Science 229: 1202.
(1985); Oi et al., BioTechniques 4: 214 (1986).
Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 17196; Morrison et al., EP 173494; Neube
rger et al., WO 8601533; Robinson et al., WO 870267.
1; Boulianne et al., Nature 312: 643 (1984); Ne.
(see Uberger et al., Nature 314: 268 (1985)).
.

Example 11 Production of Secreted Protein for High Throughput Screening Assay
The following protocol produces a supernatant containing the polypeptide to be tested. This supernatant can be used in the screening assays described herein.

First, poly-D-lysine (644 587 Boehringer-Man
nheim) stock solution (1 mg / ml in PBS) 1 in PBS (17-516F Biowhitetaker without calcium or magnesium).
Dilute to 20 to a working solution of 50 μg / ml. 200 μl of this solution
Add to each well (24 well plate) and incubate at room temperature for 20 minutes. Ensure that the solution is distributed across each well (Note: a 12-channel pipettor can be used with tips on every other channel).
Aspirate the poly-D-lysine solution and rinse with 1 ml PBS (phosphate buffered saline). PBS should remain in the wells just prior to plating the cells, and the plates can be pre-coated with polylysine up to two weeks ago.

293T cells (which do not carry cells past P + 20) at 2 × 10 ⁵ cells / well, 0.5 ml DMEM (Dulbecco Modified Eagle Medium) (4.
5G / L glucose and L-glutamine (12-604F Biowhit
10% heat-inactivated FBS (14-503F Biowhi)
ttaker) / 1 × Penstrep (17-602E Biowhite)
ker). Cells are grown overnight.

The next day, 300 μl Lipofectamine (1
8324-012 Gibco / BRL) and 5 ml Optimem I (
31985070 Gibco / BRL) / 96 well plates are mixed together. Using a small volume multichannel pipettor, about 2 μg of the expression vector containing the polynucleotide insert produced by the method described in Example 8 or 9 was placed in an appropriately labeled 96 well round bottom plate. Aliquot.
Using a multichannel pipettor, 50 μl of Lipofectamine / Opt
Add the imem I mixture to each well. Mix gently by pipetting up and down. Incubate at room temperature for 15-45 minutes. After about 20 minutes
Add 150 μl Optimem I to each well using a multichannel pipettor. As a control, one plate of vector DNA lacking the insert should be transfected with each set of transfections.

Preferably, transfection involves the following tasks in a tag team (
tag-teaming). By forming a tag team,
The time effort is halved and the cells do not spend much time on PBS. First, Mr. A sucks and removes the medium from four 24-well plates of cells, and then Mr. B rinses each well with 0.5-1 ml of PBS. Mr. A then aspirates off the PBS rinse, and Mr. B uses a 12-channel pipetter with a tip for every other channel, using 200 μl of DNA.
The / Lipofectamine / Optimem I complex is added to each row on the 24-well plate, first to the odd wells and then to the even wells. Incubate for 6 hours at 37 ° C.

While incubating the cells, either 1% BSA in DMEM with 1 × penstrep or CHO-5 medium (116.6 mg / L
Of CaCl ₂ (anhydride); 0.00130 mg / L CuSO ₄ -5H ₂ O;
050 mg / L Fe (NO ₃ ) ₃ -9H ₂ O; 0.417 mg / L FeSO ₄ −
7H ₂ O; 311.80 mg / L KCl; 28.64 mg / L MgCl ₂ ; 4
8.84 mg / L MgSO ₄ ; 6995.50 mg / L NaCl; 2400
. 0 mg / L NaHCO ₃ in; 62.50mg / L of _{NaH 2 PO 4 -H 2 O;} 71
. 02 mg / L Na ₂ HPO ₄ ; 0.4320 mg / L ZnSO ₄ -7H ₂ O;
0.002 mg / L arachidonic acid; 1.022 mg / L cholesterol; 0
. 070 mg / L DL-α-tocopherol acetate; 0.0520 mg / L linoleic acid; 0.010 mg / L linolenic acid; 0.010 mg / L myristic acid; 0.010 mg / L oleic acid; 0.010 mg / L palmitic oleic acid; 0.010 mg / L palmitic acid; 1
00 mg / L Pluronic F-68; 0.010 mg / L stearic acid; 2.20 mg / L Tween 80; 4551 mg / L D-glucose;
130.85 mg / ml L-alanine; 147.50 mg / ml L-arginine-HCl; 7.50 mg / ml L-asparagine-H ₂ O; 6.65 mg
/ Ml L-aspartic acid; 29.56 mg / ml L-cystine 2HCl-
H ₂ O; 31.29 mg / ml L-cystine-2HCl; 7.35 mg / ml
L-glutamic acid; 365.0 mg / ml L-glutamine; 18.75 mg
/ Ml glycine; 52.48 mg / ml L-histidine-HCl-H ₂ O;
106.97 mg / ml L-isoleucine; 111.45 mg / ml L-leucine; 163.75 mg / ml L-lysine HCl; 32.34 mg / ml L-methionine; 68.48 mg / ml L-phenylalanine ; 40.0 mg
/ Ml L-proline; 26.25 mg / ml L-serine; 101.05 mg
91./ml L-threonine; 19.22 mg / ml L-tryptophan;
79 mg / ml L-tyrosine-2Na-2H ₂ O; 9
9.65 mg / ml L-valine; 0.0035 mg / L biotin; 3.24
mg / L D-Ca pantothenic acid; 11.78 mg / L choline chloride; 4.65
15.60 mg / L i-inositol; 3.02 mg / L niacinamide; 3.00 mg / L pyridoxal HCl; 0.031 mg
/ L pyridoxine HCl; 0.319 mg / L riboflavin; 3.17 mg
/ L thiamine HCl; 0.365 mg / L thymidine; and 0.680 mg
/ L vitamin B ₁₂ ; 25 mM HEPES buffer; 2.39 mg / L Na hypoxanthine; 0.105 mg / L lipoic acid; 0.081 mg / L putrescine sodium-2HCl; 55.0 mg / L pyruvin Sodium acid; 0.006
7 mg / L sodium selenite; 20 μM ethanolamine; 0.122
mg-L ferric citrate; methyl-B-cyclodextrin complexed with 41.70 mg / L linoleic acid; methyl-B-cyclodextrin complexed with 33.33 mg / L oleic acid; As well as any suitable medium (methyl-B-cyclodextrin complexed with 10 mg / L retinal). (For 10% BSA stock solution, BSA (81-0
68-3 Bayer) 100 g was dissolved). The medium is filtered and 50 μl
Are collected in a 15 ml polystyrene conical for endotoxin assay.

The transfection reaction is terminated at the end of the incubation period, preferably in teams. Mr. A aspirates the transfection medium while Mr. B adds 1.5 ml of the appropriate medium to each well. Incubate at 37 ° C for 45 or 72 hours depending on the medium used:
45% for 1% BSA or 72 hours for CHO-5.

On the fourth day, 600 μl using a 300 μl multichannel pipettor
Aliquot into a 1 ml deep well plate and 2 ml of the remaining supernatant.
Aliquot into deep bottom wells. The supernatant from each well was then used for Examples 13-2.
Can be used in the assay described in 0.

If activity is obtained in any of the assays described below using the supernatant, this activity may be directly from the polypeptide (eg, as a secreted protein) or
It is particularly understood that either from or by a polypeptide that induces the expression of other proteins (the protein is then secreted into the supernatant).
Thus, the present invention further provides a method for identifying proteins in the supernatant that are characterized by activity in a particular assay.

Example 12: Construction of GAS reporter construct
One signaling pathway involved in cell differentiation and proliferation is the Jaks-ST.
Called the AT path. Proteins activated in the Jaks-STAT pathway bind to the gamma activation site “GAS” element or interferon-sensitive response element (“ISRE”), located in the promoters of many genes.
Protein binding to these elements alters the expression of the associated gene.

GAS and ISRE elements are recognized by transcriptional signal transducers and activators, a class of transcription factors called “STAT”. There are six members in the STAT family. S
tat1 and Stat3 are present in many cell types, similar to Stat2 (so that the response to IFNα is extensive). Stat4 is more limited and is not present in many cell types, but has been found in T helper class I cells after treatment with IL-12. Stat5, originally called breast growth factor, has been found at higher concentrations in other cells, including bone marrow cells. It can be activated in tissue culture cells by many cytokines.

STATs are activated and translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase (“Jaks”) family. Jaks is representative of a unique family of soluble tyrosine kinases and includes Tyk2, Jak1, Jak2, and Jak3.
These kinases show significant sequence similarity and are generally catalytically inactive in resting cells.

Jaks is activated by a wide range of receptors as summarized by the following table. (Schidler and Darnell, Ann. Rev. Bi
ochem. 64: 621-51 (1995). Jaks
The cytokine receptor family that can activate is divided into two groups:
(A) Class 1 is IL-2, IL-3, IL-4, IL-6, IL-7, IL
-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CS
Including receptors for F, GM-CSF, LIF, CNTF, and thrombopoietin; and (b) class 2 includes IFN-a, IFN-g, and IL-
10 is included. Class 1 receptors are conserved cysteine motifs (a set of four conserved cysteines and one tryptophan), and WSXWS
The motif (the membrane proximity region encoding Trp-Ser-XXX-Trp-Ser (SEQ ID NO: 2)) is shared.

Thus, upon binding of the ligand to the receptor, Jaks is activated, which in turn activates STAT, which then migrates and binds to the GAS element. This entire process is encompassed by the Jaks-STAT signaling pathway.

Therefore, Jak reflected by the binding of GAS or ISRE elements
Activation of the s-STAT pathway can be used to indicate proteins involved in cell proliferation and differentiation. For example, growth factors and cytokines are
It is known to activate the TAT pathway. (See the table below). Thus, by using a GAS element linked to a reporter molecule, Jak
Activators of the s-STAT pathway can be identified.

実施例１３〜１４に記載される生物学的アッセイにおいて使用されるプロモー
ターエレメントを含む合成ＧＡＳを構築するために、ＰＣＲに基づいたストラテ
ジーを用いてＧＡＳ−ＳＶ４０プロモーター配列を生成する。５’プライマーは
、ＩＲＦ１プロモーターにおいて見出され、そして一定の範囲のサイトカインで
の誘導の際にＳＴＡＴに結合することが以前に実証されたＧＡＳ結合部位の４つ
の直列のコピーを含むが（Ｒｏｔｈｍａｎら、Ｉｍｍｕｎｉｔｙ １：４５７−
４６８（１９９４））、他のＧＡＳエレメントまたはＩＳＲＥエレメントを代わ
りに使用し得る。５’プライマーはまた、ＳＶ４０初期プロモーター配列に対し
て相補的な１８ｂｐの配列も含み、そしてＸｈｏＩ部位に隣接する。５’プライ
マーの配列は以下である：
５’：ＧＣＧＣＣＴＣＧＡＧＡＴＴＴＣＣＣＣＧＡＡＡＴＣＴＡＧＡＴＴＴＣＣ
ＣＣＧＡＡＡＴＧＡＴＴＴＣＣＣＣＧＡＡＡＴＧＡＴＴＴＣＣＣＣＧＡＡＡＴＡ
ＴＣＴＧＣＣＡＴＣＴＣＡＡＴＴＡＧ：３’（配列番号３）。

In order to construct a synthetic GAS containing promoter elements used in the biological assays described in Examples 13-14, a GAS-SV40 promoter sequence is generated using a PCR-based strategy. The 5 ′ primer contains four tandem copies of the GAS binding site found in the IRF1 promoter and previously demonstrated to bind to STAT upon induction with a range of cytokines (Rothman et al. , Immunity 1: 457-
468 (1994)), other GAS elements or ISRE elements may be used instead. The 5 'primer also contains an 18 bp sequence complementary to the SV40 early promoter sequence and is flanked by XhoI sites. The sequence of the 5 ′ primer is:
5 ': GCGCCTCGAGATTCCCCGAAATCTAGATTTC
CCGAAATGATTTCCCCCGAAATGATTTCCCCCGAAATA
TCTGCCATCTCAATTAG: 3 ′ (SEQ ID NO: 3).

The downstream primer is complementary to the SV40 promoter and Hin
d flanking the III site: 5 ': GCGGCAAGCTTTTTGCAAAG
CCTAGGC: 3 ′ (SEQ ID NO: 4).

PCR amplification is performed using the SV40 promoter template present in the B-gal: promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI / Hind III and subcloned into BLSK2- (Stratagene). Sequencing with forward and reverse primers confirms that the insert contains the following sequence:
5 ': CTCGAG ATTCCCCGAAATCTAGATTCCCCGA
AATGATTCCCCGAAATGATTCCCCGAAATATCTG
CCATCTCAATTTAGTCAGCAACCATATAGTCCCGCCCCT
AACTCCGCCCCATCCCCCCCCTAACTCCGCCCAGTTC
CGCCCATTCTCCCGCCCCATGGCTGAACTAATTTTTTTT
TATTTATGCAGAGGGCGCAGGCCGCCTCCGGCCTCTGA
GCTATTCCGAAAGTAGTGGAGAGGCTTTTTTGGAGG
CCTAGGCTTTTGCAAA AAGCTT: 3 '(SEQ ID NO: 5).

When this GAS promoter element is bound to the SV40 promoter,
The GAS: SEAP2 reporter construct is then manipulated. Here, the reporter molecule is secreted alkaline phosphatase, or “SEAP”. Clearly, however, any reporter molecule in this or any other example can be substituted for SEAP. Well known reporter molecules that can be used in place of SEAP include chloramphenicol acetyltransferase (C
AT), luciferase, alkaline phosphatase, β-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.

Synthetic GAS-SV40 promoter elements confirmed by the above sequences were used to construct HindIII and XhoI to generate GAS-SEAP vectors.
Is used to effectively replace the SV40 promoter with the amplified GAS: SV40 promoter element and subcloned into the pSEAP-promoter vector obtained from Clontech. However, this vector does not contain a neomycin resistance gene and is therefore not preferred for mammalian expression systems.

Thus, to create a mammalian stable cell line that expresses the GAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using SalI and NotI and the GAS-SEAP / Neo vector is generated. In order to do so, these restriction sites in the multicloning site are used to make a backbone vector containing a neomycin resistance gene (eg, pGFP-1 (C
lontech)). Once this vector is transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding as described in Examples 13-14.

Other constructs can be made using the above description and replacing the GAS with a different promoter sequence. For example, the construction of a reporter molecule comprising NFK-B and EGR promoter sequences is described in Examples 15 and 16. However, many other promoters can be replaced using the protocols described in these examples. For example, SRE, IL-2, NFAT, or osteocalcin promoter alone or in combination (eg, GAS / NF-KB / EGR, G
AS / NF-KB, Il-2 / NFAT, or NF-KB / GAS) substitution. Similarly, other cell lines (eg, HELA (epithelial cells), HUVEC (endothelial cells), Reh (B cells), Saos-2 (osteoblasts), HUVAC (aortic cells), or cardiomyocytes) can be used as reporters. It can be used to test the activity of the construct.

Example 13: High Throughput Screening Assay for T Cell Activity
The following protocol is used to assess T cell activity by identifying factors and determining whether a supernatant containing a polypeptide of the invention will proliferate and / or differentiate T cells. . GA produced in Example 12 for T cell activity
Evaluate using the S / SEAP / Neo construct. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signaling pathway.
The T cells used in this assay were Jurkat T cells (ATCC accession number T
IB-152), but Molt-3 cells (ATCC accession number CRL-155).
2) and Molt-4 cells (ATCC accession number CRL-1582) cells may also be used.

Jurkat T cells are lymphoblastic CD4 + Th1 helper cells. In order to generate a stable cell line, approximately 2 million Jurkat cells were
Using IE-C (Life Technologies), GAS-SEAP
Transfect with the / neo vector (transfection procedure described below). Transfected cells are seeded at a density of approximately 20,000 cells / well and transfectants that are resistant to 1 mg / ml geneticin are selected. Growing resistant colonies, then
Test for their response to increasing concentrations of interferon gamma. The dose response of selected clones is shown.

Specifically, the following protocol provides sufficient cells for 75 wells containing 200 μl of cells. Thus, to produce sufficient cells for multiple 96-well plates, this is either scaled up or performed in multiple. Jurkat cells were treated with RPMI + 10% serum and 1% Pen
-Maintain in Strep. 2.5 ml OPTI-MEM in a T25 flask
(Life Technologies) and 10 μg of plasmid DNA. Add 2.5 ml OPTI-MEM containing 50 μl DMRIE-C and incubate at room temperature for 15-45 minutes.

During the incubation period, the cell concentration is counted, the required number of cells (10 ⁷ per transfection) is spun down, and a final concentration of 10 ⁷ cells / m
Resuspend in OPTI-MEM to l. Then 1 ml of 1 × 10 ⁷ cells in OPTI-MEM is added to the T25 flask and incubated at 37 ° C. for 6 hours. After incubation, 10 ml RPMI + 15% serum is added.

Jurkat: GAS-SEAP stable reporter strain with RPMI + 10% serum,
Maintain in 1 mg / ml geneticin and 1% Pen-Strep.
These cells are treated with a supernatant containing a polypeptide of the invention and / or induced with a polypeptide of the invention to be produced by the protocol described in Example 11.

Cells should be washed on the day of treatment with the supernatant and 500,00 per ml
Should be resuspended in fresh RPMI + 10% serum to a density of 0 cells. The exact number of cells required depends on the number of supernatants screened.
For one 96-well plate, approximately 10 million cells (100 million cells for 10 plates) are required.

To dispense cells into a 96 well dish using a 12-channel pipette, transfer the cells to a triangular reservoir boat. 200 μl of cells are transferred to each well using a 12-channel pipette (thus 1 per well)
Add 00,000 cells).

After seeding all plates, 50 μl of the supernatant is transferred directly from the 96-well plate containing the supernatant to each well using a 12-channel pipette. further,
The dose of exogenous interferon gamma (0.1, 1.0, 10 ng)
9. Add to well H10 and well H11 and use as additional positive control for the assay.

Place 96-well dishes containing Jurkat cells treated with supernatant in incubator for 48 hours (Note: this time can vary between 48-72 hours)
. Then 35 μl of sample from each well is transferred to an opaque 96 well plate using a 12 channel pipette. The opaque plate should be covered (using a cellophane® cover) and the SEA described in Example 17
It should be stored at −20 ° C. until the P assay is performed. Plates containing the remaining treated cells are placed at 4 ° C. and serve as a source of material to repeat the assay on specific wells, if desired.

As a positive control, 100 units / ml interferon gamma can be used, which is known to activate Jurkat T cells. Typically, more than 30-fold induction is observed in positive control wells.

The protocol described above can be used to make both transient and stable transfected cells, as will be apparent to those skilled in the art.

Example 14: High Throughput Screening Assay to Identify Myeloid Activity
The following protocol is used to assess myeloid activity by determining whether a polypeptide of the present invention will proliferate and / or differentiate myeloid cells. Myeloid cell activity is assessed using the GAS / SEAP / Neo construct produced in Example 12. Thus, the factor that increases SEAP activity is Jaks-S.
FIG. 6 shows the ability to activate the TATS signaling pathway. As the myeloid cells used in this assay, U937 (which is a pre-monocyte cell line), TF-1, HL60, or KG1 can be used.

In order to transiently transfect U937 cells with the GAS / SEAP / Neo construct produced in Example 12, the DEAE-Dextran method (
Kharbanda et al., 1994, Cell Growth & Differ
entry, 5: 259-265). First, 2 × 10e ⁷ U937 cells are harvested and washed with PBS. U937 cells usually have 1
Grow in RPMI 1640 medium containing 10% heat inactivated fetal bovine serum (FBS) supplemented with 00 units / ml penicillin and 100 mg / ml streptomycin.

Next, 0.5 mg / ml DEAE-Dextran, 8 μg GAS-SE
AP2 plasmid DNA, 140 mM NaCl, 5 mM KCl, 375 μM
Na ₂ HPO ₄ .7H ₂ O, 1 mM MgCl ₂ , and 675 μM CaCl ₂
Suspend cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer containing Incubate at 37 ° C for 45 minutes.

Cells were washed with RPMI 1640 medium containing 10% FBS and then 10
Resuspend in ml complete medium and incubate at 37 ° C. for 36 hours.

GAS-SEAP / U937 stable cells are obtained by growing the cells in 400 μg / ml G418. G418-free medium is used to grow routinely, but cells are transferred at 400 μg / ml G418 every two months for two passages.
Should be regrowth in.

These cells are tested by collecting 1 × 10 ⁸ cells (which is sufficient for 10 96-well plate assays) and washed with PBS. Suspend cells in the above 200 ml growth medium at a final density of 5 × 10 ⁵ cells / ml. Plate 200 μl of cells per well (ie 1 × 10 ⁵ cells / well) in a 96 well plate.

Add 50 μl of the supernatant prepared by the protocol described in Example 11. Incubate at 37 ° C for 48-72 hours. As a positive control, 1
00 units / ml of interferon gamma can be used, which is known to activate U937 cells. Typically, over 30-fold induction is observed in positive control wells. The supernatant is SE according to the protocol described in Example 17.
AP assay.

Example 15: High Throughput Screening Assay to Identify Neuronal Activity
As cells undergo differentiation and proliferation, a group of genes is activated through many different signaling pathways. One of these genes, EGR1 (early growth response gene 1), is induced in various tissues and cell types upon activation. EG
The R1 promoter is responsible for such induction. EGR bound to reporter molecule
One promoter can be used to assess cell activation.

Specifically, the following protocol is used to assess neuronal activity in the PC12 cell line. PC12 cells (Phenochrom)
cells) proliferate and / or differentiate upon activation with many mitogens (eg, TPA (tetradecanoylphorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth factor)). Is known. During this treatment, EGR1 gene expression is activated. Thus, PC12 cell activation can be assessed by stably transfecting PC12 cells with a construct comprising an EGR promoter linked to a SEAP reporter.

The EGR / SEAP reporter construct can be assembled by the following protocol.
EGR-1 promoter sequence (-633- + 1) (Sakamoto K et al., O
ncogene 6: 867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers:
5'GCCGCTCGAGGGGATGACAGCGATAGAACCCCGG-
3 ′ (SEQ ID NO: 6)
5'GCGAAGCTTCCGCGACTCCCCGGATCCGCTCC-3
'(SEQ ID NO: 7).

The EGR1 amplification product can then be inserted into this vector using the GAS: SEAP / Neo vector created in Example 12. Restriction enzyme XhoI / H
Using indIII to linearize the GAS: SEAP / Neo vector,
Remove the S / SV40 stuffer. The same enzyme is used to restrict the EGR1 amplification product. Ligate the vector and the EGR1 promoter.

To prepare a 96 well plate for cell culture, a coating solution (
Collagen type I (Upstate Biotech Inc. Catalog No. 08)
-115) diluted 1:30 with 30% ethanol (sterile filtration)
Add 2 ml per plate, or 50 ml per well of a 96 well plate and allow to air dry for 2 hours.

PC12 cells were pre-coated on 10 cm tissue culture dishes supplemented with penicillin (100 units / ml) and streptomycin (100 μg / ml), 10% horse serum (JRH BIOSCIENCES, catalog number 124).
49-78P) RPMI-164 with 5% heat inactivated fetal bovine serum (FBS)
Grow conventionally in 0 medium (Bio Whittaker). Split from 1 to 4 every 3-4 days. Peel off the cells by scraping the cells,
Then re-suspend by pipetting up and down more than 15 times.

The EGR / SEAP / Neo construct was assembled from Lipofecta as described in Example 11.
Transfect PC12 using the mine protocol. EGR-S
EAP / PC12 stable cells are obtained by growing the cells in 300 μg / ml G418. Medium without G418 is used for routine growth, but cells should be regrown in 300 μg / ml G418 for 2 passages every 1-2 months.

To assay for neuronal activity, 10 cm plates with approximately 70-80% confluent cells are screened by removing old media. Wash once with PBS (phosphate buffer solution). The cells were then cultured in low serum medium (RPMI with antibiotics and 1% horse serum and 0.5% FBS).
-1640) overnight in the cells.

The next morning, remove the media and wash the cells with PBS. Scrape the cells from the plate and suspend the cells in 2 ml of low serum medium. The cell number is counted and additional low serum medium is added to reach a final cell density of 5 × 10 ⁵ cells / ml.

200 μl of cell suspension is added to each well of a 96 well plate (1 × 1
Equivalent to ⁵ cells / well). 50 μl of the supernatant produced according to Example 11
Add at 37 ° C. for 48-72 hours. As a positive control, P via EGR
Growth factors known to activate C12 cells (eg, 50 ng / μl nerve growth factor (NGF)) can be used. Typically, more than 50-fold SEAP induction is seen in positive control wells. According to Example 17, the supernatant was
AP assay.

Example 16: High Throughput Screening Assay for T Cell Activity
NF-κB (Nuclear Factor κB) is induced by exposure to LPS or thrombin by a wide variety of drugs, including inflammatory cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-α and lymphotoxin-β. As well as transcription factors activated by the expression of specific viral gene products. As a transcription factor, NF-κB expresses genes involved in immune cell activation, controls apoptosis (NF-κB appears to protect cells from apoptosis), B and T cell development, antiviral Modulates responses and antimicrobial responses, as well as multiple stress responses.

In unstimulated conditions, NF-κB is retained in the cytoplasm with I-κB (inhibitor κB). However, upon stimulation, I-κB is phosphorylated and degraded, causing a nuclear shuttle of NF-κB, thereby activating transcription of the target gene. Examples of target genes activated by NF-κB include IL-2, IL-6, GM-CSF, ICAM-1 and MHC class 1.

Due to its central role and ability to respond to a range of stimuli, NF-
A reporter construct utilizing the κB promoter element is used to screen the supernatant produced in Example 11. An activator or inhibitor of NF-κB is useful for the treatment of disease. For example, inhibitors of NF-κB may be used to treat diseases associated with acute or chronic NF-κB activation (eg, rheumatoid arthritis).

To construct a vector containing the NF-κB promoter element, PCR
Use a strategy based on. The upstream primer has an NF-κB binding site (
GGGACTTTTCCC) (SEQ ID NO: 8), four tandem copies, containing an 18 bp sequence complementary to the 5 'end of the SV40 early promoter sequence and adjacent to the XhoI site:
5 ': GCGGCCTCGAGGGGACTTTCCCCGGGGACTTTCC
GGGGACTTTCCGGGATTTCCATCCTGGCCATCTCAA
TTAG: 3 '(SEQ ID NO: 9).

The downstream primer is complementary to the 3 ′ end of the SV40 promoter;
And adjacent to the HindIII site:
5 ': GCGGCAAGCTTTTTGCAAAGCCTAGGC: 3' (SEQ ID NO: 4).

PCR amplification is performed using the SV40 promoter template present in the pB-gal: promoter plasmid obtained from Clontech.
The resulting PCR fragment is digested with XhoI and HindIII and subcloned into BLSK2- (Stratagene). T7 and T
Confirm that the insert contains the following sequence by sequencing with 3 primers:
5 ': CTCGAGGGGACTTTCCCCGGGGACTTTCCGGGGGA
CTTTCCGGGAACTTTCCATCTGCCATCTCAATTTAGTC
AGCAACCATAGTCCCCCCCCTAACTCCGCCCCATCCCC
GCCCCTAACTCCGCCCAGTTCCGCCCATTTCCCCCC
CCATGGCTGAACTAATTTTTTTTATTTAGCAGAGGC
CGAGGCCGCCCTCGGCCTCTGAGCATTTCCAGAAGTA
GTGAGGAGGCTTTTTGGAGGCCTAGGGCTTTGCAA
AAAGCTT: 3 ′ (SEQ ID NO: 10).

XhoI and HindIII are then used to replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) with this NF-κB / SV40 fragment. However, this vector does not contain a neomycin resistance gene and is therefore not preferred for mammalian expression systems.

In order to generate a stable mammalian cell line, the NF-κB / SV40 / SEAP cassette was used with the restriction enzymes SalI and NotI as described above for NF-κB / SEA.
Remove from the P vector and insert into a vector containing neomycin resistance.
Specifically, after restriction treatment of pGFP-1 with SalI and NotI, NF-
The κB / SV40 / SEAP cassette was inserted into pGFP-1 (Clontech) to replace the GFP gene.

Once the NF-κB / SV40 / SEAP / Neo vector was generated, stable Jurkat T cells were generated according to the protocol described in Example 13,
And maintain. Similarly, a method for assaying supernatants containing these stable Jurkat T cells is also described in Example 13. As a positive control, exogenous TNFα (0.1, 1, 10 ng) is added to wells H9, H10, and H11 (typically wells where 5-10 fold activation is observed).

Example 17: Assay for SEAP activity
SE as a reporter molecule for the assays described in Examples 13-16
AP activity is determined by Tropix Phospho-li according to the following general procedure.
Assay using ght Kit (Cat. No. BP-400). Troop
ix Phospho-light Kit is a dilution buffer used in the following:
An assay buffer and a reaction buffer are provided.

Fill the dispenser with 2.5 × Dilution Buffer and dispense 15 μl of 2.5 × Dilution Buffer to an Optiplate containing 35 μl of supernatant. Seal the plate with a plastic sealer and incubate at 65 ° C. for 30 minutes. Keep the Optiplate apart to avoid uneven heating.

Cool the sample to room temperature for 15 minutes. Empty the dispenser and fill with assay buffer. Add 50 μl assay buffer and incubate for 5 minutes at room temperature. Empty the dispenser and fill the reaction buffer (see table below). Add 50 μl of reaction buffer and incubate for 20 minutes at room temperature. The intensity of the chemiluminescent signal is time dependent, and it takes about 10 minutes to read 5 plates with a luminometer, so 5 plates are processed at a time, and the second set after 10 minutes. Should start.

Read the relative light unit in the luminometer. Set H12 as blank and print the result. An increase in chemiluminescence indicates reporter activity.

（実施例１８：低分子の濃度および膜透過性における変化を同定するハイスル
ープットスクリーニングアッセイ）
レセプターへのリガンドの結合は、カルシウム、カリウム、ナトリウムのよう
な低分子およびｐＨの細胞内レベルを変化させ、ならびに膜電位を変化させるこ
とは公知である。これらの変化を特定細胞のレセプターに結合する上清の同定を
行うアッセイで測定し得る。以下のプロトコルは、カルシウムについてのアッセ
イを記載するが、このプロトコルは、カリウム、ナトリウム、ｐＨ、膜電位、ま
たは蛍光プローブにより検出可能な任意の他の低分子における変化を検出するよ
うに容易に改変され得る。

Example 18: High-throughput screening assay to identify changes in small molecule concentration and membrane permeability
Ligand binding to receptors is known to alter intracellular levels of small molecules such as calcium, potassium, sodium and pH, as well as membrane potential. These changes can be measured in assays that identify supernatants that bind to receptors on specific cells. The following protocol describes an assay for calcium, but this protocol is easily modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule detectable by a fluorescent probe. Can be done.

The following assay measures changes in fluorescent molecules that bind small molecules (Molecular Probes) using a fluorometric imaging plate reader ("FLIPR"). Obviously, any fluorescent molecule that detects a small molecule is a calcium fluorescent molecule, fluo-4 (Molecular Probes) as used herein.
, Inc. Catalog number F-14202).

For adherent cells, seed cells at 10,000-20,000 cells / well in a Co-star black 96 well plate with clear bottom. Plate with CO ₂
Incubate for 20 hours in incubator. Adherent cells were washed in a Biotek washer with 200 μl HBSS (Hank's Balanced Salt
Wash twice with Solution and leave 100 μl of buffer after the last wash.

A stock solution of 1 mg / ml fluo-4 was added to 10% pluronic acid (pluro
nic acid) DMSO. To load cells with fluo-4,
12 μg / ml fluo-4 (50 μl) is added to each well. The plate is incubated for 60 minutes at 37 ° C. in a CO ₂ incubator. The plate is washed 4 times with HBSS in a Biotek washer leaving 100 μl of buffer.

For non-adherent cells, the cells are spun down from the culture medium. 50 cells
Resuspend to 2-5 × 10 ⁶ cells / ml with HBSS in a ml conical tube. F containing 10% Pluronic acid DMSO solution per 1 ml of cell suspension
Add 4 μl of luo-4 (1 mg / ml). The tube is then placed in a 37 ° C. water bath for 30-60 minutes. Cells are washed twice with HBSS and 1 × 10 ⁶ cells / ml
And resuspend in microplates at 100 μl / well. Centrifuge the plate at 1000 rpm for 5 minutes. Next, put the plate in Denley
After washing once with 200 μl in CellWash, the final volume is brought to 100 μl by a suction step.

For non-cell based assays, each well contains a fluorescent molecule such as fluo-4. Supernatant is added to the wells and fluorescence changes are detected.

To measure intracellular calcium fluorescence, FLIPR is set for the following parameters: (1) System gain is 300-800 mW; (2)
Exposure time is 0.4 seconds; (3) Camera F / Stop is F / 2;
(4) Excitation is 488 nm; (5) Fluorescence is 530 nm; and (6)
Sample addition is 50 μl. The increase in fluorescence at 530 nm is due to intracellular Ca
⁺⁺ indicates an extracellular signaling event that results in an increase in concentration.

Example 19: High Throughput Screening Assay to Identify Tyrosine Kinase Activity
Protein tyrosine kinase (PTK) refers to a diverse group of transmembrane and cytoplasmic kinases. Receptor protein tyrosine kinase (RPTK)
) A range of mitogenics, including PDGF, FGF, EGF, NGF, HGF and insulin receptor subfamily within the group
And receptors for metabolic growth factors. In addition, there is a large RPTK family whose corresponding ligands are unknown. RPTK ligands include primarily secreted low molecular weight proteins, but also include membrane-bound and extracellular matrix proteins.

Activation of RPTK by ligand involves ligand-mediated receptor dimerization, which results in transphosphorylation of receptor subunits and activation of cytoplasmic tyrosine kinases. Cytoplasmic tyrosine kinases include the src family (
Examples include receptor-related tyrosine kinases of src, yes, lck, lyn, fyn), and non-receptor-bound and cellular substrate protein tyrosine kinases such as the Jak family. These members are cytokine superfamily (eg, interleukin, interferon, GM-
Mediates signal transduction induced by receptors of CSF and leptin).

Because of the wide variety of known factors that can stimulate tyrosine kinase activity, it is interesting to identify these novel human secreted proteins that can activate tyrosine kinase signaling pathways. Therefore, the following protocol is designed to identify novel human secreted proteins that can activate the tyrosine kinase signaling pathway.

Target cells (eg, primary keratinocytes) were purchased from Nalge Nunc (Naperville, IL) at a density of approximately 25,000 cells / well 96
Seed well Loprodyne Silent Screen Plate. Plates are sterilized by rinsing twice with 100% ethanol for 30 minutes, rinsed with water, and then dried overnight. Some plates were treated with 100 ml cell culture grade type I collagen (50 mg / ml), gelatin (2%) or polylysine (
50 mg / ml) (all of these are Sigma Chemicals (St. L
ouis, MO)) or Becton Dickinso
Coat with 10% Matrigel purchased from n (Bedford, MA) or calf serum for 2 hours, rinse with PBS, and store at 4 ° C. Seed 5,000 cells / well containing growth medium and the manufacturer Alamar Biosc
ienses, Inc. (Sacramento, CA) as described, a
By indirectly quantifying the cell number after 48 hours using lamarBlue,
Assay cell proliferation on these plates. Becton Dickins
On (Bedford, MA) Falcon plate cover # 3071 is used to cover the Loprodyne Silent Screen Plate. F
The alcon Microtest III cell culture plate can also be used in some proliferation experiments.

To prepare the extract, A431 cells are seeded on a nylon membrane of Loprodyne plates (20,000 / 200 ml / well) and cultured overnight in complete medium. Cells are incubated in serum-free basal medium for 24 hours to rest. EGF (60 ng / ml) or 50 μl of the supernatant produced in Example 11 with 5-2
After treatment for 0 minutes, the medium was removed and 100 ml of extraction buffer (20 mM HEP
ES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.
1% SDS, 2 mM Na ₃ VO ₄ , 2 mM Na ₄ P ₂ O ₇ and Boeheri
A mixture of protease inhibitors (# 1836170)) obtained from nger Mannheim (Indianapolis, Ind.)) is added to each well and the plate is shaken on a rotary shaker at 4 ° C. for 5 minutes. The plate is then transferred to a vacuum transfer manifold.
old) and filter the extract through a 0.45 mm membrane at the bottom of each well using house vacuum. The extract is collected in a 96 well capture / assay plate at the bottom of the vacuum manifold and immediately placed on ice. To obtain a clarified extract by centrifugation, after solubilizing with a surfactant for 5 minutes, the contents of each well are removed and centrifuged at 16,000 × g for 15 minutes at 4 ° C.

The filtered extract is tested for the level of tyrosine kinase activity. A number of methods for detecting tyrosine kinase activity are known, but one method is described herein.

In general, the tyrosine kinase activity of the supernatant is assessed by determining the ability to phosphorylate tyrosine residues on a particular substrate (biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (cell division kinase cd
c2-p34 corresponding to amino acids 6 to 20) and PSK2 (corresponding to amino acids 1 to 17 of gastrin). Both peptides are substrates for a series of tyrosine kinases and are available from Boehringer Mannheim.

The tyrosine kinase reaction is set up by adding the following components in order. First, 10 μl of 5 μM biotinylated peptide was added, and then ATP / Mg ²⁺ (
10 μl of 5 mM ATP / 50 mM MgCl ₂ , 5 × assay buffer (40
mM imidazole hydrochloride, pH 7.3, 40 mM β-glycerophosphate, 1 mM
EGTA, 100 mM MgCl ₂ , 5 mM MnCl ₂ , 0.5 mg / ml
Add 10 μl BSA), 5 μl sodium vanadate (1 mM) and finally 5 μl water. The components are mixed gently and the reaction mixture is preincubated at 30 ° C. for 2 minutes. Add 10 μl of control enzyme or filtered supernatant to start the reaction.

The tyrosine kinase assay reaction is then stopped by adding 10 μl of 120 mM EDTA and the reaction is placed on ice.

Tyrosine kinase activity is measured by transferring a 50 μl aliquot of the reaction mixture to a microtiter plate (MTP) module and incubating at 37 ° C. for 20 minutes. As a result, streptavidin (streptava)
din) Associate coated 96-well plate with biotinylated peptide.
Wash the MTP module 4 times with 300 μl / well PBS. Next, anti-phosphotyrosine conjugated to horseradish peroxidase (phosphopotyro)
Sine) 75 μl of antibody (anti-P-Tyr-POD (0.5 u / ml)) is added to each well and then incubated at 37 ° C. for 1 hour. The wells are washed as above.

Next, a peroxidase substrate solution (Boehringer Mannheim)
) Add 100 μl and incubate at room temperature for at least 5 minutes (up to 30 minutes). The absorbance of the sample at 405 nm is measured using an ELISA reader. The level of bound peroxidase activity is quantified using an ELISA reader, which reflects the level of tyrosine kinase activity.

Example 20: High Throughput Screening Assay to Identify Phosphorylation Activity
Also used as a possible alternative and / or complement of the protein tyrosine kinase activity assay described in Example 19 is an assay that detects the activation (phosphorylation) of key intracellular signaling intermediates Can do.
For example, as described below, one particular assay is Erk-1 and Erk-2.
Kinase tyrosine phosphorylation can be detected. However, Raf, JNK, p38
Phosphorylation of MAP, Map Kinase Kinase (MEK), MEK Kinase, Src, Muscle Specific Kinase (MuSK), other molecules such as IRAK, Tec and Janus, as well as any other phosphoserine, phosphotyrosine or phosphothreonine molecule In these assays, Erk-1 or E
It can be detected by replacing rk-2.

Specifically, the wells of a 96-well ELISA plate were washed with protein G (1 μg /
ml) Coat 0.1 ml for 2 hours at room temperature (RT) to make the assay plate. The plates are then rinsed with PBS and blocked with 3% BSA / PBS for 1 hour at RT. Next, protein G plates were Erk-1 and Erk.
Two commercially available monoclonal antibodies against rk-2 (100 ng / well) (S
ant cruz biotechnology) (1 hour at RT). (To detect other molecules, this process can be easily modified by exchanging monoclonal antibodies that detect any of the above molecules). After rinsing 3-5 times with PBS, store plates at 4 ° C. until use.

A431 cells are seeded in 96-well Loprodyne filter plates at 20,000 / well and cultured overnight in growth medium. The cells are then basal medium (D
EGF (6 ng / well) or Example 1 after starving in MEM) for 48 hours
Treat with 50 μl of the supernatant obtained in 1 for 5-20 minutes. The cells are then solubilized and the extract is filtered and placed directly into the assay plate.

After 1 hour incubation with the extract at RT, the wells are rinsed again. Commercial MAP kinase preparation (10 ng / well) as a positive control
Is used in place of the A431 extract. Next, the plate is moved to Erk-1 and Erk.
Treat with commercially available polyclonal (rabbit) antibody (1 μg / ml) that specifically recognizes the phosphorylated epitope of rk-2 kinase (1 hour at RT). This antibody is biotinylated by standard procedures. The bound polyclonal antibody is then combined with Europium streptavidin and Europium fluorescence enhancer in Wallac D
Quantify by continuous incubation (time-resolved fluorescence) in an ELFIA instrument. An increase in fluorescent signal over background indicates phosphorylation.

(Example 21: Method for determining a change in a gene corresponding to a polynucleotide)
RNA isolated from the entire family or individual patient presenting the desired phenotype (eg, disease) is isolated. CDNA is then generated from these RNA samples using protocols known in the art (see Sambrook).
Next, this cDNA is used as a template for PCR using a primer surrounding the region of interest in SEQ ID NO: X. Suggested PCR conditions are Si
using the buffer solution described in Dransky et al. , Science 252: 706 (1991), consisting of 35 cycles of 95 ° C. for 30 seconds; 52-58 ° C. for 60-120 seconds; and 70 ° C. for 60-120 seconds.

Next, the PCR product was categorized into SequiTherm Polymerase (Epi
center Technologies) and sequencing using a primer labeled at its 5 ′ end with T4 polynucleotide kinase.
The intron-exon boundary of the selected exon is also determined and the genomic PCR product is analyzed to confirm the result. Next, the PCR product having the suspicious mutation is cloned and sequenced, and the direct sequencing result is confirmed.

PCR products were obtained from Holton et al., Nucleic Acids Research.
ch, 19: 1156 (1991) and cloned into a T-tail vector and T7 polymerase (United States Biochemica).
Sequence using l). Affected individuals are identified by mutations that are not present in unaffected individuals.

Genomic rearrangements are also observed as a way to determine changes in genes corresponding to polynucleotides. Genomic clones isolated according to Example 2 were digoxigenin deoxy-uridine 5′-triphosphate (Boehringer Man
heim) and Johnson et al., M
methods Cell Biol. 35: FISH is performed as described in 73-99 (1991). Hybridization with a labeled probe is performed using a large excess of human cot-1 DNA for specific hybridization to the corresponding genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidol and propidium iodide to produce a combination of C and R bands. Aligned images for accurate mapping can be generated using a triple band filter set (Chroma Tec).
hnology, Brattleboro, VT) and cooled charge coupled device camera (
Photometrics, Tucson, AZ) and a variable excitation wavelength filter (Johnson et al., Genet. Anal. Tech. Appl., 8: 7).
5 (1991)). ISee Graphical
Program System (Invision Corporation)
on, Durham, NC) using image acquisition, analysis and chromosome fractionation (f
racional ) Length measurement is performed. Chromosomal changes in the genomic region to which the probe hybridizes are identified as insertions, deletions and translocations. These changes are used as diagnostic markers for related diseases.

Example 22 Method for Detecting Abnormal Levels of Polypeptides in Biological Samples
A polypeptide of the invention can be detected in a biological sample, and if an increase or decrease in the polypeptide level is detected, the polypeptide is a marker for a specific phenotype. It will be appreciated that there are numerous detection methods and, therefore, those skilled in the art can modify the following assays to suit their particular needs.

For example, an antibody sandwich ELISA is used to detect a polypeptide in a sample (preferably in a biological sample). The wells of the microtiter plate are coated with specific antibodies at a final concentration of 0.2-10 μg / ml. This antibody is either monoclonal or polyclonal and is
Produced by the method described in 0. The well is blocked so that non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated with the sample containing the polypeptide for more than 2 hours at RT. Preferably, results should be confirmed using serial dilutions of the sample. The plate is then washed 3 times with deionized or distilled water to remove unbound polypeptide.

Next, 50 μl of specific antibody-alkaline phosphatase conjugate is added to 25-400 n
Add at a concentration of g and incubate at room temperature for 2 hours. The plate is again washed three times with deionized or distilled water to remove unbound conjugate.

75 μl of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution is added to each well and incubated for 1 hour at room temperature. This reaction is measured with a microtiter plate reader.
A standard curve is generated using serial dilutions of the control sample and the polypeptide concentration is plotted on the X axis (log scale) and the fluorescence or absorbance is plotted on the Y axis (uniform scale). A standard curve is used to interpolate the polypeptide concentration in the sample.

(Example 23: Prescription)
The invention also provides for a disease or disorder (eg, such as any one or more of the diseases or disorders disclosed herein) by administering to a subject an effective amount of a therapeutic agent.
A method of treating and / or preventing is provided. A polynucleotide or polypeptide (including fragments and variants), agonists or antagonists thereof, and / or antibodies thereto, in combination with a pharmaceutically acceptable carrier type (eg, sterile carrier) by a therapeutic agent Is meant.

The therapeutic agent is considered a good medical practice, taking into account the individual patient's clinical condition (especially the side effects of the therapeutic agent alone treatment), delivery site, method of administration, dosing regimen and other factors known to those skilled in the art. ) In a manner that complies with Therefore, the target “effective amount” in the present specification is determined by taking such consideration into consideration.

As a general suggestion, the total pharmaceutically effective amount of therapeutic agent administered parenterally per dose is in the range of about 1 μg / kg / day to 10 mg / kg / day of patient weight,
As noted above, this is left to therapeutic discretion. More preferably, for this hormone, this dose is at least 0.01 mg / kg / day, most preferably between about 0.01 mg / kg / day and about 1 mg / kg / day for humans. When administered continuously, typically the therapeutic agent is about 1 μg / kg / hour to about 50 μg / kg.
Administer either 1-4 injections per day at a dosing rate / hour or by continuous subcutaneous infusion (eg, using a minipump). Intravenous bag solutions may also be used. The duration of treatment necessary to observe the change and the interval after treatment where the response occurs are:
It seems to change depending on the desired effect.

Therapeutic agents are administered orally, rectally, parenterally, intracisternally.
y) may be administered intravaginally, intraperitoneally, topically (such as by powder, ointment, gel, drop, or transdermal patch), buccal or oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulant or any type of formulation adjuvant. As used herein, the term “
“Parenteral” refers to modes of administration including intravenous and intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.

The therapeutic agents of the present invention are also suitably administered by a sustained release system. Suitable examples of sustained release therapeutic agents are oral, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, topical (such as by powder, ointment, gel, infusion, or transdermal patch), Administer by mouth, orally or as a nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulant or any type of formulation adjuvant. As used herein, the term “parenteral” means intravenous, intramuscular,
Refers to modes of administration including intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.

The therapeutic agents of the present invention are also suitably administered by a sustained release system. Suitable examples of sustained release therapeutic agents include suitable polymeric materials, such as, for example, semipermeable polymer matrices in the form of film or microcapsule moldings, suitable hydrophobic materials (
For example, as an emulsion in an acceptable oil) or ion exchange resin, and poorly soluble derivatives (such as poorly soluble salts).

Sustained release matrices include polylactide (US Pat. No. 3,773,919).
No., EP 58,481), a copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers 22: 547-5.
56 (1983)), poly (2-hydroxyethyl methacrylate) (Lang
er et al. Biomed. Mater. Res. 15: 167-277 (19
81), and Langer, Chem. Tech. 12: 98-105 (19
82)), ethylene vinyl acetate (Langer et al., Ibid.) Or poly-D
-(-)-3-hydroxybutyric acid (EP133,988).

Sustained release therapeutic agents also include therapeutic agents of the present invention encapsulated in liposomes (generally, Langer, Science 249: 1527-1533 (199).
0); Treat et al., Liposomes in the Therapy o
f Infectious Disease and Cancer, Loop
z-Berstain and Fiddler (ed.), Liss, New Yor
k, pages 317-327 and pages 353-365 (1989)). Liposomes containing therapeutic agents are prepared by methods known per se: D
E3, 218, 121; Epstein et al., Proc. Natl. Acad. S
ci. (USA) 82: 3688-3692 (1985); Hwang et al., Pr.
oc. Natl. Acad. Sci. (USA) 77: 4030-4034 (1
980); EP52,322; EP36,676; EP88,046; EP14
EP 142,641; Japanese Patent Application No. 83-118008; US Pat. Nos. 4,485,045 and 4,544,545; and EP
No. 102,324. Usually, liposomes are small (about 200-800 cm) unilamellar type, where the lipid content is greater than about 30 mol% cholesterol and the selected proportion is adjusted for optimal therapeutic agents. .

In still further embodiments, the therapeutic agents of the invention are delivered by a pump (Langer, supra; Sefton, CRC Crit. Ref. Biom).
ed. Eng. 14: 201 (1987); Buchwald et al., Surger.
y 88: 507 (1980); Saudek et al. Engl. J. et al. Med.
321: 574 (1989)).

Other controlled release systems are described by Langer (Science 249: 1527-15.
33 (1990)).

For parenteral administration, in one embodiment, in general, the therapeutic agent is toxic to the recipient in the desired degree of purity, in a pharmaceutically acceptable carrier, i.e. the dosage and concentration used. It is formulated by mixing in a unit dosage injectable form (solution, suspension or emulsion) with one that is not and compatible with the other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to therapeutic agents.

Generally, formulations are prepared by contacting the therapeutic agent uniformly and intimately with liquid carriers or finely divided solid carriers or both. Next, if necessary, the product is shaped into the desired formulation. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Nonaqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes.

The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are not toxic to recipients at the dosages and concentrations used, such as phosphates, citrates, succinates, acetic acids and other organic acids or their salts. Buffering agents; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides (eg, polyarginine or tripeptides); proteins such as serum albumin, gelatin or immunoglobulins; polyvinylpyrrolidone Hydrophilic polymers such as: amino acids such as glycine, glutamic acid, aspartic acid or arginine; monosaccharides, disaccharides and other carbohydrates including cellulose or derivatives thereof, glucose, mannose or dextrin; chelating agents such as EDTA Sugar sugars such as mannitol or sorbitol Lumpur; counterions such as sodium; and / or polysorbate include nonionic surfactants such as poloxamers or PEG.

The therapeutic agent is typically about 0.1 mg / ml to 100 mg / ml, preferably 1
Formulated in such vehicles at a concentration of -10 mg / ml and a pH of about 3-8. It is understood that by using the specific excipients, carriers or stabilizers described above, polypeptide salts are formed.

Any pharmaceutical agent used for therapeutic administration can be sterile. Aseptic conditions are easily achieved by filtration through sterile filtration membranes (eg 0.2 micron membranes). In general, the therapeutic agent is placed in a container having a sterile access port, for example, an intravenous solution bag or vial with a stopper puncturable with a hypodermic needle.

The therapeutic agents are typically stored in unit dose or multi-dose containers, such as sealed ampoules or vials, as an aqueous solution or lyophilized formulation for reconstitution.
As an example of a lyophilized formulation, 1% (w / v) sterile filtered into a 10 ml vial.
) Fill with 5 ml of aqueous therapeutic solution and lyophilize the resulting mixture. The lyophilized therapeutic agent is reconstituted with bacteriostatic water for injection to prepare an infusion solution.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the therapeutic agent of the present invention. A notice of the form established by a government agency that regulates the manufacture, use or sale of a medicinal product or biological product may be attached to such a container, and this notice may be attached to the government relating to manufacture, use or sale for human administration. Represents institutional approval. In addition, the therapeutic agent may be used in combination with other therapeutic compounds.

The therapeutic agents of the present invention can be administered alone or in combination with an adjuvant. Adjuvants that can be administered with the therapeutic agents of the present invention include, but are not limited to: alum, alum + deoxycholic acid (I
mmunoAg), MTP-PE (Biocine Corp.), QS21 (
Genentech, Inc. ), BCG, and MPL. In certain embodiments, the therapeutic agents of the invention are administered in combination with alum. In another specific embodiment, the therapeutic agent of the invention is administered in combination with QS-21. Additional adjuvants that can be administered with the therapeutic agents of the present invention include, but are not limited to: monophosphoryl lipid immunomodulators, AdjuV.
ax 100a, QS-21, QS-18, CRL1005, aluminum salt,
MF-59, and Virosomal adjuvant technology. Vaccines that can be administered with the therapeutic agents of the present invention include, but are not limited to: MMR (measles, mumps, rubella), polio, chickenpox, tetanus / diphtheria, A Hepatitis B, hepatitis B, influenza B, whooping cough
oping cow), pneumonia, influenza, Lyme disease, rotavirus,
Cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,
Vaccines directed against protection against typhoid and pertussis. The combinations can be administered, for example, either as a mixture simultaneously, separately but simultaneously or in parallel; or sequentially. This includes instructions that the combined drugs are administered together as a therapeutic mixture, and the procedure where the combined drugs are administered separately but simultaneously (eg, through separate intravenous lines to the same individual) Including. “Combination” administration further includes separately administering one of the compounds or agents given first, followed by the second.

The therapeutic agents of the invention can be administered alone or in combination with other therapeutic agents. Therapeutic agents that can be administered with the therapeutic agents of the present invention include, but are not limited to: other members of the TNF family, chemotherapeutic agents,
Antibiotics, steroidal and non-steroidal anti-inflammatory agents, traditional immunotherapy agents,
Cytokines and / or growth factors. The combinations can be administered, for example, either as a mixture simultaneously, separately but simultaneously or in parallel; or sequentially. This includes instructions that the combined agents are administered together as a therapeutic mixture, and also includes a procedure where the combined agents are administered separately but simultaneously (eg, through separate intravenous lines to the same individual). Also includes. "Combination" administration is one of the compounds or drugs given first, followed by the second.
Further comprising administering one separately.

In one embodiment, the therapeutic agents of the invention are administered in combination with a TNF family member. TNF, TNF that can be administered with the therapeutic agent of the present invention
Related or TNF-like molecules include, but are not limited to: soluble forms of TNF-α, lymphotoxin-α, (LT-α, TNF-β
Also known as), LT-β (found in complex heterotrimers LT-α2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1B
BL, DcR3, OX40L, TNF-γ (International Publication No. WO96 / 14328)
), AIM-I (International Publication No. WO 97/33899), Endokine (end
okine) -α (International Publication No. WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG, and Neutrokine (neutrok)
ine) -α (International Publication No. WO 98/18921), OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD4
0 and 4-IBB, TR2 (International Publication No. WO96 / 34095), DR3 (
International publication number WO 97/33904), DR4 (international publication number WO 98/328)
56), TR5 (International Publication Number WO98 / 30693), TR6 (International Publication Number WO98 / 30694), TR7 (International Publication Number WO98 / 41629), TR
ANK, TR9 (International Publication Number WO98 / 56892), TR10 (International Publication Number WO98 / 54202), 312C2 (International Publication Number WO98 / 06842)
, And TR12, and soluble forms of CD154, CD70, and CD1
53.

In certain embodiments, the therapeutic agents of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and / or protease inhibitors. As a nucleoside reverse transcriptase inhibitor that can be administered in combination with the therapeutic agent of the present invention,
Examples include, but are not limited to: RETROVIR ^™ (zidovudine / AZT), VIDEOX ^™ (didanocin / ddI), HIVID ^™ (zarcitabine / ddC), ZERTI ^™ (stavudine / d4T), EPIVIR ^™ (lamivudine / 3TC) , And COMBIVIR ^™ (zidovudine / lamivudine). Non-nucleoside reverse transcriptase inhibitors that can be administered in combination with the therapeutic agents of the present invention include, but are not limited to: VIRAMUNE ^™ (
Nevirapine), RESCRIPTOR ^™ (Delavirdin), and SUSTIV
A ^TM (Efavi lens). Protease inhibitors that can be administered in combination with the therapeutic agents of the present invention include, but are not limited to: CR
IXIVAN ^™ (indinavir), NORVIR ^™ (
Ritonavir), INVIRASE ^™ (Saquinavir (sa
quinavir)), and VIRACEPT ^™ (nelfinavir (nelf)
inavir)). In certain embodiments, the antiretroviral agent, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, and / or protease inhibitor is for treating AIDS and / or for preventing or treating HIV infection, It can be used in any combination with the therapeutic agent of the present invention.

In other embodiments, the therapeutic agents of the invention can be administered in combination with an anti-opportunistic infection agent. Anti-opportunistic agents that can be administered in combination with the therapeutic agents of the present invention include, but are not limited to: TRIMETHOP
RIM-SULFAMETHOXAZOLE ^™ , DAPSONE ^™ , PENTA
MIDINE ^TM , ATOVAQUEONE ^TM , ISONIAZID ^TM , RIFAM
PIN ^TM , PYRAZINAMIDE ^TM , ETHAMBUTOL ^TM , RIFAB
UTIN ^™ , CLARITHROMYCIN ^™ , AZITROMYCIN ^™ ,
GANICLOVIR ^™ , FOSCARNET ^™ , CIDOFOVIR ^™ , F
LUCONAZOLE ^TM , ITRACONAZOLE ^TM , KETOCONAZO
LE ^TM , ACYCLOVIR ^TM , FAMCICOLVIR ^TM , PYRIMETH
AMINE ^™ , LEUCOVORIN ^™ , NEPOGEN ^™ (filgrastim / G-CSF), and LEUKINE ^™ (sargramostim / GM-CSF). In certain embodiments, the therapeutic agent of the invention is an opportunistic Pneumocystis carinii.
TRIMETHOPRIM-S for the prophylactic treatment or prevention of pneumonia infection
ULFAMETHOXAZOLE ^™ , DAPSONE ^™ , PENTAMIDIN
Used in any combination with E ^™ and / or ATOVAQUEONE ^™ . In another specific embodiment, the therapeutic agent of the present invention is an opportunistic Mycobact.
In order to prevent or treat erium avium complex infection prophylactically, ISO
NIAZID ^™ , RIFAMPIN ^™ , PYRAZINAMIDE ^™ , and / or
Or used in any combination with ETHAMBUTOL ^™ . In another specific embodiment, the therapeutic agent of the present invention is an opportunistic Mycobacterium t
RIFAB for the prophylactic treatment or prevention of beruculosis infection
UTIN ^™ , CLARITHROMYCIN ^™ , and / or AZITHRO
Used in any combination with MYCIN ^™ . In another specific embodiment, the therapeutic agents of the invention are used in any combination with GANCILOVIR ^™ , FOSCARNET ^™ , and / or CIDOFOVIR ^™ to prevent or treat opportunistic cytomegalovirus infections prophylactically. The In another specific embodiment, the therapeutic agent of the present invention comprises FLUCONAZOLE ^™ , ITRACONAZOLE ^™ , and / or for prophylactically treating or preventing opportunistic fungal infections.
Or used in any combination with KETOCONAZOLE ^™ . In another specific embodiment, the therapeutic agent of the present invention provides ACYCLOV for prophylactic treatment or prevention of opportunistic herpes simplex virus type I and / or type II infection.
Used in any combination with IR ^™ and / or FAMCICOLVIR ^™ . In another specific embodiment, the therapeutic agent of the present invention is an opportunistic Toxopla.
To prevent or prevent sma gondii infection, PYRIME
Used in any combination with THAMINE ^™ and / or LEUCOVORIN ^™ . In another specific embodiment, the therapeutic agents of the invention are used in any combination with LEUCOVORIN ^™ and / or NEPOGEN ^™ to prevent or treat opportunistic bacterial infections prophylactically.

In a further embodiment, the therapeutic agent of the invention is administered in combination with an antiviral agent. Antiviral agents that can be administered with the therapeutic agents of the present invention include acyclovir, ribavirin, amantadine, and remantidine (reman
tidin), but is not limited thereto.

In a further embodiment, the therapeutic agent of the invention is administered with an antibiotic. Antibiotics that can be administered with the therapeutic agent of the present invention include amoxicillin, β
-Lactamase, aminoglycoside, β-lactam (glycopeptide), β-lactamase, clindamycin, chloramphenicol, cephalosporin, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolone, macrolide, metronidazole, Examples include, but are not limited to, penicillin, quinolone, rifampin, streptomycin, sulfonamide, tetracycline, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.

Conventional non-specific immunosuppressive agents that can be administered with the therapeutic agents of the present invention include steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin (15 -Deoxyspergualin), and other immunosuppressive agents that act by inhibiting the function of the T cell response, but are not limited to these.

In certain embodiments, the therapeutic agents of the invention can be administered in combination with immunosuppressive agents. An immunosuppressant preparation that can be administered with the therapeutic agent of the present invention is ORTHO.
CLONE ^TM (OKT3), SANDIMMUNE ^TM / NEORAL ^TM / SAN
GDYA ^™ (Cyclosporine), PROGRAF ^™ (Tacrolimus), CELL
CEPT ^™ (microphenolate), azathioprine, glycoticosteroid,
And RAPAMUNE ^™ (sirolimus). In certain embodiments, immunosuppressive agents can be used to prevent organ or bone marrow transplant rejection.

In further embodiments, the therapeutic agents of the invention are administered alone or in combination with one or more intravenous immunoglobulin preparations. Intravenous immunoglobulin preparations that can be administered in combination with the therapeutic agents of the present invention include GAMLAR ^™ , IVEE.
Examples include, but are not limited to, GAM ^™ , SANDOGLOBULIN ^™ , GAMMAGARD S / D ^™, and GAMIMUNE ^™ . In certain embodiments, the therapeutic agents of the invention are administered in combination with intravenous immunoglobulin preparations in transplantation therapy (eg, bone marrow transplantation).

In further embodiments, the therapeutic agents of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that can be administered with the therapeutic agents of the present invention include glucocorticoids and non-steroidal anti-inflammatory agents, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones , Salicylic acid derivatives, thiazinecarboxamide, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzidoamine, bucolome, difenpyramide, ditazole, emorphazone, guaiazulene , Nabumetone, nimesulide, orgothein, oxaceptol, paraniline, perizoxal, pifoxime, prochiazone, proxazole, and tenidap But, but it is not limited to these.

In another embodiment, the composition of the invention is administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that can be administered with the therapeutic agents of the present invention include antibiotic derivatives (eg, doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (eg, tamoxifen); antimetabolites (eg,
Fluorouracil, 5-FU, methotrexate, Phloxuridine (flo
xuridine), interferon α-2b, glutamic acid, prikamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (eg, carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, Hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate)
Hormones (eg medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestol diphosphate, chlorotrianicene, and test lactone); nitrogen mustard derivatives (Eg, mephalene, chlorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (eg, betamethasone sodium phosphate); and others (eg, dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate,
And etoposide), but are not limited thereto.

In certain embodiments, the therapeutic agents of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or in any combination of components of CHOP. In another embodiment, the therapeutic agent of the invention is administered in combination with Rituximab. In a further embodiment, the therapeutic agent of the present invention comprises Rituxmab and CHOP.
Or with any combination of Rituxmab and CHOP components.

In further embodiments, the therapeutic agents of the invention are administered in combination with cytokines. Cytokines that can be administered with the therapeutic agents of the present invention include IL2
IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13,
IL15, anti-CD40, CD40L, IFN-γ and TNF-α include, but are not limited to. In another embodiment, the therapeutic agent of the present invention comprises any interleukin (IL-1α, IL-1β, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11
IL-12, IL-13, IL-14, IL-15, IL-16, IL-17
, IL-18, IL-19, IL-20 and IL-21).

In a further embodiment, the therapeutic agent of the invention is administered in combination with an angiogenic protein. Angiogenic proteins that can be administered with the therapeutic agents of the present invention include the glioma derived growth factor (GD) as disclosed in European Patent EP-399816.
GF); platelet-derived growth factor as disclosed in European Patent EP-682110
A (PDGF-A); platelet-derived growth factor-B (PDGF-B) as disclosed in European Patent EP-282317; placental growth factor (PIGF) as disclosed in International Publication No. WO 92/06194; Hauser et al., Gorwth Fa
ctors 4: placenta growth factor-2 (PIGF-2) as disclosed in 259-268 (1993); vascular endothelial growth factor (VEGF) as disclosed in WO 90/13649; European patent Vascular endothelial growth factor-A (VEGF-A) as disclosed in EP-506477; International Publication No. WO 96/3951
Vascular endothelial growth factor-2 (VEGF-2) as disclosed in No. 5; Vascular endothelial growth factor-B (VEGF-3); Vascular endothelial growth factor B as disclosed in International Publication No. WO 96/26736 -186 (VEGF-B186); International Publication Number WO
Vascular endothelial growth factor-D (VEGF-D) as disclosed in 98/02543
Vascular endothelial growth factor as disclosed in WO 98/07832
D (VEGF-D); and vascular endothelial growth factor-E (VEGF-E) as disclosed in German Patent DE19639601. The above references are incorporated herein by reference.

In a further embodiment, the therapeutic agent of the invention is administered in combination with a hematopoietic growth factor. Hematopoietic growth factors that can be administered with the therapeutic agent of the present invention include LEU.
KINE ^™ (SARGRAMOSTIM ^™ ) and NEPOGEN ^™ (FIL
GRASTIM ^™ ), but is not limited to these.

In a further embodiment, the therapeutic agents of the invention are administered in combination with fibroblast growth factor. Examples of fibroblast growth factors that can be administered with the therapeutic agent of the present invention include FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, and FGF.
-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, F
Examples include, but are not limited to, GF-12, FGF-13, FGF-14, and FGF-15.

In further embodiments, the therapeutic agents of the invention are administered in combination with other therapeutic or prophylactic regimes (eg, radiation therapy).

Example 24 Method for Treating Reduced Polypeptide Levels
The present invention relates to a method for treating an individual in need of increasing the level of a polypeptide of the invention in the body, the method comprising a therapeutically effective amount of an agonist of the invention (including a polypeptide of the invention). Administering a composition comprising it to such an individual. It will further be appreciated that conditions caused by a decrease in the standard or normal expression level of secreted proteins in an individual can be treated by administering the polypeptides of the present invention, preferably in secreted form. Thus, the present invention also provides a method of treating an individual in need of increased levels of polypeptide. The method includes administering to such an individual a therapeutic agent comprising an amount of the polypeptide that increases the activity level of the polypeptide in such an individual.

For example, patients with reduced levels of polypeptide take the polypeptide for 6 consecutive days at a daily dose of 0.1-100 μg / kg. Preferably the polypeptide is in secreted form. Exact details of the dosing regimen based on administration and formulation can be found in Example 2
3 is provided.

Example 25: Method for treating elevated levels of polypeptide
The invention also relates to a method for treating an individual in need of reducing the level of a polypeptide of the invention in the body, the method comprising a therapeutically effective amount of an antagonist of the invention (polypeptide and antibody of the invention). Administration) to such an individual.

In one example, antisense technology is used to inhibit production of a polypeptide of the invention. This technique is one example of a method for reducing the level of polypeptides, preferably in secreted form, resulting from a variety of etiologies such as cancer.
For example, in patients diagnosed with abnormally elevated levels of polypeptide, antisense polynucleotides can be administered at 0.5, 1.0, 1.5, 2.0 and 3.0 mg /
Administered intravenously at 21 kg / day for 21 days. If you have enough resistance to this treatment,
This treatment is repeated after a 7-day withdrawal period. Antisense polynucleotide formulations are provided in Example 23.

Example 26: Treatment method using gene therapy-ex vivo
One method of gene therapy is to transplant fibroblasts that can express the polypeptide into a patient. In general, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue culture medium and divided into small pieces. Place a lump of tissue on the wet surface of the tissue culture flask and place approximately 10 pieces in each flask. Invert the flask, close it tightly, and leave it at room temperature overnight. After 24 hours at room temperature, the flask is inverted, the tissue mass remains fixed at the bottom of the flask, and fresh medium (eg, Ha containing 10% FBS, penicillin and streptomycin) is used.
m F12 medium). The flask is then incubated at 37 ° C. for about 1 week.

At this point, fresh medium is added and then replaced every few days. After two more weeks of culture, monolayer fibroblasts appear. Trypsinize this monolayer,
Scale up to a larger flask.

PMV-7 (Kirsc) flanked by Moloney murine sarcoma virus long terminal repeats
hmeier, P.M. T.A. Et al., DNA, 7: 219-25 (1988)).
After digestion with RI and HindIII, treatment with calf intestinal phosphatase.
The linear vector is fractionated on an agarose gel and purified using glass beads.

CDNAs encoding the polypeptides of the invention correspond to the 5 'and 3' end sequences, respectively, described in Example 1, using primers and having appropriate restriction sites and start / stop codons, if necessary. PCR primers can be used to amplify. Preferably, the 5 ′ primer contains an EcoRI site and the 3 ′ primer contains a HindIII site. Equal amounts of Moloney murine sarcoma virus linear backbone and amplified EcoRI and HindIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for ligating the two fragments. The ligation mixture is then used to transform bacterial HB101. It is then plated on agar containing kanamycin to confirm that the vector has the gene of interest correctly inserted.

Amphotropic pA317 or GP + am12 packaging cells
Grow to confluent density in tissue culture in Dulbecco's Modified Eagle Medium (DMEM) containing 0% calf serum (CS), penicillin and streptomycin. Next, an MSV vector containing the gene of interest is added to the medium and the packaging cells are transduced with this vector. At this time, the packaging cell produces infectious virus particles containing the gene (herein, the packaging cell is referred to as a producer cell).

Fresh medium is added to the transduced producer cells, which is then harvested from confluent producer cells in 10 cm plates. After the spent medium containing infectious virus particles is filtered through a Millipore filter to remove peeled producer cells, this medium is used to infect fibroblasts. The medium is removed from the subconfluent plate of fibroblasts and quickly replaced with medium from the producer cells. This medium is removed and replaced with fresh medium. If the titer of the virus is high, virtually all fibroblasts are infected and no selection is necessary. If the titer is very low, it is necessary to use a retroviral vector with a selectable marker such as neo or his. Once the fibroblasts are efficiently infected, the fibroblasts are analyzed to determine if protein is being produced.

The engineered fibroblasts are then transplanted into the host, either alone or after growing confluent on cytodex 3 microcarrier beads.

Example 27: Gene therapy using an endogenous gene corresponding to the polynucleotide of the present invention
Another method of gene therapy according to the present invention provides for endogenous polynucleotide sequences of the present invention, eg, US Pat. No. 5,641,670 (issued June 24, 1997);
International publication number WO 96/29411 (published September 26, 1996); International publication number WO 94/12650 (published August 4, 1994); Koller et al., P.
roc. Natl. Acad. Sci. USA, 86: 8932-8935 (1
989); and Zijlstra et al., Nature, 342: 435-438.
(1989) includes operably binding to a promoter via homologous recombination. This method involves the activation of a gene that is present in the target cell but not expressed in the cell or expressed at a lower level than desired.

A polynucleotide construct comprising a promoter and a targeting sequence is generated. This target sequence is homologous to the 5 'non-coding sequence of the endogenous polynucleotide sequence adjacent to the promoter. This targeting sequence is 5 ′ of the polynucleotide sequence.
Close enough to the ends, so that this promoter is operably linked to this endogenous sequence during homologous recombination. This promoter and targeting sequence is
Can be used to amplify. Preferably, the amplified promoter contains different restriction enzyme sites on the 5 'and 3' ends. Preferably, the 3 ′ end of the first targeting sequence comprises the same restriction enzyme site as the 5 ′ end of the amplified promoter, and the 5 ′ end of the second targeting sequence is the 3 ′ end of the amplified promoter. Contains the same restriction sites.

The amplified promoter and amplified targeting sequence are digested with appropriate restriction enzymes and subsequently treated with bovine intestinal phosphatase. The digested promoter and digested targeting sequence are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for ligation of these two fragments. This construct is size fractionated on an agarose gel and then purified by phenol extraction and ethanol precipitation.

In this example, the polynucleotide construct is administered as a naked polynucleotide via electroporation. However, the polynucleotide construct can also be administered with transfection facilitating agents (eg, liposomes, viral sequences, viral particles, precipitants, etc.). Such methods of delivery are known in the art.

Once the cell is transfected, homologous recombination occurs and the promoter is operably linked to the endogenous polynucleotide sequence. This results in the expression of a polynucleotide corresponding to this polynucleotide in the cell. Expression can be detected by immunological staining or any other method known in the art.

Fibroblasts are obtained from the subject by skin biopsy. The resulting tissue was treated with DMEM +
Place in 10% fetal bovine serum. Logarithmic or early stationary phase fibroblasts are trypsinized and rinsed in nutrient medium from the plastic surface. An aliquot of the cell suspension is removed for counting and the remaining cells are subjected to centrifugation. The supernatant was aspirated and the pellet was washed with 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM K
CI, 0.7 mM Na ₂ HPO ₄ , 6 mM dextrose). The cells are recentrifuged, the supernatant is aspirated, and the cells are resuspended in electroporation buffer containing 1 mg / ml acetylated bovine serum albumin. This final cell suspension contains about 3 × 10 ⁶ cells / ml. Electroporation should be performed immediately after resuspension.

Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to a locus corresponding to a polynucleotide of the present invention, plasmid pUC18 (MBI Fermentas, Amherst, NY
) Is digested with HindIII. The CMV promoter is amplified by PCR with an XbaI site at the 5 ′ end and a BamHI site at the 3 ′ end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a HindIII site at the 5 ′ end and an XbaI site at the 3 ′ end; the other non-coding sequence (fragment) 2) is amplified with a BamHI site at the 5 ′ end and a HindIII site at the 3 ′ end. The CMV promoter and fragments thereof (1 and 2) are combined with the appropriate enzymes (CMV promoter-XbaI and BamHI; fragment 1-XbaI; fragment 2-B
amHI) and ligated together. The resulting ligation product is HindII
Digested with I and ligated with pUC18 plasmid digested with HindIII.

Plasmid DNA was sterilized with a sterile cuvette (B
io-Rad). The final DNA concentration is generally at least 120 μ
g / ml. Then 0.5 ml of the cell suspension (containing about 1.5 × 10 ⁶ cells) is added to the cuvette and the cell suspension and DNA solution are mixed gently. Electroporation is performed using a Gene-Pulser instrument (Bio-Rad). Capacitance and voltage are set to 960 μF and 250-300 V, respectively. Increasing the voltage reduces cell survival, but dramatically increases the proportion of viable cells that stably incorporate the introduced DNA into its genome. Given these parameters, a pulse time of about 14-2
0 mSec should be observed.

The electroporated cells are maintained at room temperature for about 5 minutes, and then the contents of the cuvette are gently removed using a sterile transfer pipette. The cells are pre-warmed nutrient medium (D with 15% bovine serum in a 10 cm dish.
MEM) is added directly to 10 ml and incubated at 37 ° C. The next day, the medium is aspirated and replaced with 10 ml of fresh medium and an additional 16-24
Incubate for hours.

The engineered fibroblasts are then injected into the host, either alone or after being grown to confluence on cytodex 3 microcarrier beads. here,
This fibroblast produces a protein product. The fibroblasts can then be introduced into a patient as described above.

Example 28: Treatment method using gene therapy-in vivo
Another aspect of the invention is the use of in vivo gene therapy methods to treat disorders, diseases, and conditions. This gene therapy method involves the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or antisense RNA) sequences into animals to increase or decrease polypeptide expression. A polynucleotide of the invention can be operably linked to a promoter, or any other genetic element required for expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, for example, WO 90/11092, WO 98/11779; US Pat. Nos. 5,693,622, 5,705,151, 5580859; Tabata et al. . Res. 35 (3): 470-479 (1997); Chao et al., Pharmacol. Res. 35 (6): 517-522 (1997); Wolff, Neuromuscul. Disorder. 7 (5): 314-318 (1997), Schwartz et al., Gene Ther. 3 (5): 405-411 (1996); Tsurumi et al., Circulation 94 (12): 3281-3290 (1996), incorporated herein by reference.

The polynucleotide construct can be delivered by any method that delivers an injectable substance to an animal cell, such as an injection into the interstitial space of a tissue (heart, muscle, skin, lung, liver, intestine, etc.). The polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA is any delivery vehicle that acts to assist, facilitate, or facilitate entry into the cell (such as a viral sequence, viral particle, liposomal formulation, lipofectin, or precipitant). Is included). However, the polynucleotides of the invention can also be prepared in liposomal formulations (eg, Felgner) that can be prepared by methods well known to those skilled in the art.
P. L. (1995) Ann. NY Acad. Sci. 772: 126-1
39 and Abdallah B.H. (1995) Biol. Cell 85 (
1): as taught in 1-7).

The polynucleotide vector construct used in this gene therapy method is:
Preferably, the construct is not integrated into the host genome and does not contain sequences that allow replication. Any strong promoter known to those skilled in the art can be used to drive the expression of DNA. Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transient nature of polynucleotide synthesis in those cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for a period of up to 6 months.

This polynucleotide construct is used in tissues within animals (muscle, skin, brain, lung, liver,
Spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue) Can be delivered to space. The interstitial space of this tissue can be intercellular fluid, mucopolysaccharide matrix (between reticulated fibers of organ tissues, elastic fibers in the walls of blood vessels or chambers, collagen fibers of fibrous tissues), or connective tissue sheath Includes the same substrate in sex muscle cells or in the hiatus of the bone. This is likewise the space occupied by circulating plasma and lymph fluid in the lymph channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They can be conveniently delivered by injection into a tissue containing these cells. They are preferably delivered to and expressed in differentiated, persistent non-dividing cells, but delivery and expression may be non-differentiated cells or cells that are not fully differentiated (eg, blood stem cells Or skin fibroblasts). In vivo, muscle cells are particularly qualified for their ability to take up and express polynucleotides.

For naked polynucleotide injection, an effective dosage of DNA or RNA is in the range of about 0.05 g / kg body weight to about 50 mg / kg body weight. Preferably, this dosage is from about 0.005 mg / kg to about 20 mg / kg, and more preferably from about 0.05 mg / kg to about 5 mg / kg. Of course, as those skilled in the art will appreciate, this dosage will vary according to the tissue site of the infusion. Appropriate and effective dosages of the nucleic acid sequences can be readily determined by one skilled in the art and can depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral injection route into the interstitial space of the tissue. However, other parenteral routes can also be used,
This includes, for example, inhalation of aerosol formulations for delivery to the lung or bronchial tissue, throat, or nasal mucosa, among others. Furthermore, the naked polynucleotide construct can be delivered to the artery during the angioplasty by the catheter used in this procedure.

The dose response effect of injected polynucleotide in muscle in vivo is determined as follows. Appropriate template DNA for production of mRNA encoding a polypeptide of the invention is prepared according to standard recombinant DNA methodology. Mold DN
Either A (which can be either circular or linear) is used as naked DNA or complexed with liposomes. The mouse quadriceps are then injected with various amounts of template DNA.

Five to six week old female and male Balb / C mice received 0.3 ml of 2.5%
Anesthetize by intraperitoneal injection of Avertin. A 1.5 cm incision is made in the anterior thigh and the quadriceps is directly visualized. Template DNA is injected into a 0.1 ml carrier and injected through a 27 gauge needle with a 1 cc syringe for 1 minute at a depth of about 0.2 cm into the knee about 0.5 cm from the distal insertion site of the muscle. . Suture is performed on the injection site for future positioning and the skin is closed with a stainless steel clip.

After an appropriate incubation time (eg 7 days), muscle extracts are prepared by cutting out all four heads. Every 15 μm section of each quadriceps is stained histochemically for protein expression. The time course for protein expression can be done in a similar manner, except that four animals from different mice are collected at different times. The persistence of DNA in muscle after injection can be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experiments in mice were
NA can be used to extrapolate appropriate dosages and other treatment parameters in humans and other animals.

(Example 29: Transgenic animal)
The polypeptides of the present invention can also be expressed in transgenic animals. Mouse, rat, rabbit, hamster, guinea pig, pig, miniature pig, goat,
Any species of animal, including but not limited to sheep, cows and non-human primates (eg, baboons, monkeys and chimpanzees) can be used to create transgenic animals. In certain embodiments, techniques described herein or otherwise known in the art are used for expression of the polypeptides of the invention in humans as part of a gene therapy protocol.

Any technique known in the art can be used to introduce a transgene (ie, a polynucleotide of the present invention) into an animal, and the founder line (fou) of the transgenic animal.
nder line). Such techniques are described in pronuclear microinjection (Patterson et al., Appl. Microbiol. Biotec.
hnol. 40: 691-698 (1994); Carver et al., Biotec
hnology (NY) 11: 1263-1270 (1993); Wright
Biotechnology (NY) 9: 830-834 (1991); and Hoppe et al., US Pat. No. 4,873,191 (1989)); Retrovirus-mediated gene transfer into germline (Van der Putten et al., Pr.
oc. Natl. Acad. Sci. USA 82: 6148-6152 (19
85)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompso)
n et al., Cell 56: 313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3: 180).
3-1814 (1983)); introduction of a polynucleotide of the invention using a gene gun (see, eg, Ulmer et al., Science 259: 1745 (1993)); into embryonic pluripotent stem cells Introduction of the nucleic acid construct and transfer of this stem cell into the blastocyst; and sperm-mediated gene transfer (La
vitrano et al., Cell 57: 717-723 (1989)); and the like. For a review of such techniques, see Gordon, “Transgenic An, which is incorporated herein by reference in its entirety.
imals ", Intl. Rev. Cytol. 115: 171-229 (19
89).

Any technique known in the art can be used to generate transgenic clones containing the polynucleotides of the invention (eg, derived from cultured, quiescently induced embryonic cells, fetal cells or adult cells). Nuclear transfer of enucleated oocytes into the nucleus (Campell et al., Nature 380: 64-66 (1996); Wil
mut et al., Nature 385: 810-813 (1997))).

The present invention provides transgenic animals that have the transgene in all their cells, as well as animals that have the transgene in some (but not all) cells (ie, mosaic animals or chimeras). A transgene can be a single transgene or concatamer (eg, head-to-head tandem or head-to-head).
Can be incorporated as multiple copies (such as tail tandem). This transgene is also described, for example, by the teachings of Lasko et al. (Lasko et al., Proc. Natl. Ac
ad. Sci. USA 89: 6232-6236 (1992)) and can be selectively introduced into and activated in specific cell types. The regulatory sequences required for such cell type specific activation will depend on the particular cell type of interest and will be apparent to those skilled in the art. If it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. In short, when such a technique is utilized, a vector containing several nucleotide sequences homologous to an endogenous gene is converted into a nucleotide sequence of the endogenous gene via homologous recombination with a chromosomal sequence. Designed to destroy the function of the nucleotide sequence. Transgenes can also be selectively introduced into specific cell types and thus, for example, Gu et al. (Gu et al., Science 26
5: 103-106 (1994)), the endogenous gene is inactivated only in the introduced cell type. The regulatory sequences required for such cell type specific inactivation will depend on the particular cell type of interest and will be apparent to those skilled in the art.

Once a transgenic animal has been created, the expression of the recombinant gene can be assayed using standard techniques. Initial screening can be accomplished by Southern blot analysis or PCR techniques to confirm by animal tissue analysis that transgene integration has occurred. The level of transgene mRNA expression in tissues of transgenic animals is also determined by Northern blot analysis, in situ hybridization analysis and reverse transcriptase P of tissue samples obtained from animals.
It can be assessed using techniques including but not limited to CR (rt-PCR). Samples of transgenic gene expressing tissues can also be assessed immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once founder animals are generated, they can be bred, inbred, outbred or crossed to produce a particular animal colony. Examples of such mating strategies include, but are not limited to: cross-breeding founder animals with more than one integration site to establish another lineage; additive expression of each transgene By effect, inbred lines of separate lines to generate complex transgenics that express transgenes at higher levels; both for enhanced expression and elimination of the need for animal screening by DNA analysis Crosses of heterozygous transgenic animals that generate animals homozygous for the integration site; crosses of separate homozygous lines to generate compound heterozygous or homozygous lines; and the desired experimental model Mating to place the transgene on the appropriate different background.

The transgenic animals of the present invention are effective in detailing the biological function of the polypeptides of the present invention, studying conditions and / or disorders associated with abnormal expression, and ameliorating such conditions and / or disorders. It has uses including but not limited to animal model systems useful for screening for compounds.

(Example 30: Knockout animal)
Endogenous gene expression can also be achieved using targeted homologous recombination of genes and / or
Or it can be reduced by inactivating or “knocking out” its promoter (eg, Smithies et al., Nature 317: 230-2).
34 (1985); Thomas and Capecchi, Cell 51: 5.
03-512 (1987); Thompson et al., Cell 5: 313-32.
1 (1989); each of which is incorporated herein by reference in its entirety). For example, a variant of the invention, a non-functional polynucleotide (or completely unrelated DNA) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding region or the regulatory region of this gene) Sequence) can be used with or without a selectable marker and / or a negative selectable marker to transfect cells expressing the polypeptide of the invention in vivo. In another embodiment, techniques known in the art are used to create knockouts in cells that contain the gene of interest but do not express it. Insertion of this DNA construct via targeted homologous recombination results in inactivation of the targeted gene. Such an approach is particularly suitable for research and agricultural fields that can be used to generate offspring of animals with targeted genes that are inactive in modifications to embryonic stem cells (eg, Thomas and Capecchi 1987 and Thompson 1989). , See above). However, this approach is conventional for use in humans where the recombinant DNA construct is administered directly to the required site in vivo or targeted using appropriate viral vectors apparent to those skilled in the art. Can be adapted.

In a further embodiment of the invention, cells that are genetically engineered to express a polypeptide of the invention, or cells that are genetically engineered to express no polypeptide of the invention (eg, a knockout) are treated in vivo with a patient. To be administered. Such cells can be obtained from patients (ie, animals including humans) or MHC compatible donors, and fibroblasts, bone marrow cells, blood cells (eg, lymphocytes), adipocytes, muscle cells, endothelial cells, etc. Including, but not limited to. The cells can be transformed into, for example, transduction (using viral vectors and preferably vectors that incorporate the transgene into the cell genome) or transfection procedures (plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Endogenous regulation, including, but not limited to, the introduction of a coding sequence of a polypeptide of the invention into a cell or binding to this coding sequence and / or a polypeptide of the invention. To destroy the array
Genetic engineering in vitro using recombinant DNA technology. The coding sequence of the polypeptide of the present invention may be placed under the control of a strong constitutive or inducible promoter or promoter / enhancer to achieve expression and preferably secretion of the polypeptide of the present invention. Engineered cells that express and preferably secrete the polypeptides of the invention may be systemically introduced into a patient, for example, in the circulation or intraperitoneally.

Alternatively, the cells can be incorporated into the matrix and transplanted into the body (
For example, genetically engineered fibroblasts can be transplanted as part of a skin graft; genetically engineered endothelial cells can be transplanted as part of a lymphatic or vascular graft (eg, Anderson et al., US Patent). No. 5,399,349 and Mu
See lligan and Wilson, US Pat. No. 5,460,959. Each of which is incorporated herein by reference in its entirety).

If the cells to be administered are non-self or non-MHC compatible cells, they can be administered using well-known techniques that interfere with the development of a host immune response against the introduced cells. For example, the cells can be introduced in a capsule form, which does not allow the introduced cells to be recognized by the host immune system, while allowing for the exchange of components with the immediate extracellular environment.

The transgenic and “knockout” animals of the present invention can be used to elaborate on the biological function of the polypeptides of the present invention, to study diseases, disorders and / or conditions associated with abnormal expression, and to such diseases, disorders and It has uses that include, but are not limited to, animal model systems useful in screening for compounds that are effective in ameliorating the condition.

(Example 31: Production of antibody)
(A. Hybridoma technology)
The antibodies of the present invention can be prepared by various methods. (Current Pr
autocols, see Chapter 2). As one example of such a method, cells expressing a polypeptide of the invention are administered to an animal to induce production of serum containing polyclonal antibodies. In a preferred method, a preparation of the polypeptide of the invention is prepared and purified so that it is substantially free of natural contaminants. Such preparations are then introduced into animals in order to produce higher specific activity polyclonal antisera.

Monoclonal antibodies specific for the polypeptides of the invention are prepared using hybridoma technology (Kohler et al., Nature 256: 495).
(1975); Kohler et al., Eur. J. et al. Immunol. 6: 511 (1
976); Kohler et al., Eur. J. et al. Immunol. 6: 292 (197
6); Hammerling et al .: Monoclonal Antibodies
and T-Cell Hybridomas, Elsevier, N .; Y.
563-681 (1981)). In general, an animal (preferably a mouse) is immunized with a polypeptide of the invention, or more preferably with a secretory polypeptide-expressing cell. Such polypeptide-expressing cells are preferably supplemented with 10% fetal calf serum (inactivated at about 56 ° C.) in any suitable tissue culture medium and about 10 g / l of non-essential amino acids, about 1 Cultured in Earle's modified Eagle's medium supplemented with 1,000,000 U / ml penicillin and about 100 μg / ml streptomycin.

Such mouse splenocytes are extracted and fused with an appropriate myeloma cell line. Any suitable myeloma cell line can be used in accordance with the present invention;
It is preferred to use the parent myeloma cell line (SP2O) available from CC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and then Wands et al. (Gastroenterology 80: 225-232 (
1981)) and cloned by limiting dilution. The hybridoma cells obtained by such selection are then assayed to identify clones that secrete antibodies capable of binding the polypeptides of the invention.

Alternatively, additional antibodies that can bind to the polypeptides of the invention can be generated in a two-step procedure using anti-idiotype antibodies. Such a method is
Take advantage of the fact that the antibody itself is an antigen and therefore it is possible to obtain an antibody that binds to a second antibody. According to this method, an animal (preferably a mouse) is immunized using a protein specific antibody. The splenocytes of such animals are then used to produce hybridoma cells. The hybridoma cells are then screened to identify clones that produce an antibody whose ability to bind to an antibody polypeptide specific for the protein of the invention can be blocked by the polypeptide of the invention. Such antibodies include anti-idiotypic antibodies against an antibody polypeptide specific for a protein of the invention, and immunize an animal to induce the formation of a further antibody polypeptide specific for the protein of the invention. Used for.

For in vivo use of antibodies in humans, antibodies are “humanized”. Such antibodies can be produced by using genetic constructs derived from hybridoma cells that produce the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein (for a review, see Morrison, Science 229: 120.
2 (1985); Oi et al., BioTechniques 4: 214 (1986).
); Cabilly et al., US Pat. No. 4,816,567; Taniguchi
Et al., EP 17196; Morrison et al., EP 173494; Neub
erger et al., WO 8601533; Robinson et al., WO 87026.
71; Boulianne et al., Nature 312: 643 (1984); N
See euberger et al., Nature 314: 268 (1985)).

(B) Isolation of antibody fragments directed against polypeptides from a library of scFvs)
A naturally occurring V gene isolated from human PBL is constructed into a library of antibody fragments containing reactivity to a polypeptide of the present invention that a donor may or may not have been exposed to (eg, US Pat. No. 5,885,793, which is incorporated herein by reference in its entirety).

Library rescue. A scFvs library is constructed from human PBL RNA as described in PCT Publication WO 92/01047. About 10 ⁹ E. coli harboring phagemids to rescue phage displaying antibody fragments. coli using, (containing 1% glucose and 100 [mu] g / ml ampicillin) 2 × TY of 50ml (2 × TY-AMP- GLU) was inoculated, and shaking products et O. D. Grow to 0.8 . 5 of this culture
ml was used to inoculate 50 ml of 2 × TY-AMP-GLU and 2 × 10 ⁸ TU of Δgene 3 helper (M13Δ gene III, PCT publication WO 92/01047).
) And incubate the culture for 45 minutes at 37 ° C. without shaking and then incubate for 45 minutes at 37 ° C. with shaking. This culture was incubated at 4000 r. p. m. The pellet is resuspended in 2 liters of 2 × TY (containing 100 μg / ml ampicillin and 50 μg / ml kanamycin) and grown overnight. PCT publication WO
Prepare as described in 92/01047.

M13Δ gene III is prepared as follows: M13Δ gene III helper phage does not encode the gene III protein. Therefore, phage displaying antibody fragments (phagemids) have greater binding avidity for antigen. During phage morphogenesis, infectious M13Δ gene III particles are generated by growing helper phage in cells carrying the pUC19 derivative supplying the wild type gene III protein. The culture is incubated for 1 hour at 37 ° C. without shaking and then for an additional hour at 37 ° C. with shaking. Cells are centrifuged (IEC-Centra 8,400r
. p. m. 10 μm), 100 μg / ml ampicillin and 25 μg / ml
2 × TY broth (2 × TY-AMP-KAN) 3 containing ml kanamycin
Resuspended in 00 ml and grown overnight with shaking at 37 ° C. The phage particles were purified and concentrated from the culture medium by two PEG precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS, and passed through a 0.45 μm filter (Minisart NML; Sartorius), approximately 10 ¹³
A final concentration of transducing units / ml (ampicillin resistant clones) is obtained.

Library panning. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg / ml or 10 μg / ml of a polypeptide of the invention. Tubes with 3% 2% Marvel-PBS
Block for 2 hours at 7 ° C., then wash 3 times in PBS. Approximately 10 ¹³ TU of phage is applied to the tube and incubated for 30 minutes at room temperature while tumbling up and down on a rotating disc and then allowed to stand for an additional 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS.
Phages were eluted by adding 1 ml of 100 mM triethylamine and rotating up and down on a rotating plate for 15 minutes, after which the solution was washed with 0.5 ml of 1.
Neutralize immediately with 0M Tris-HCl, pH 7.4. The eluted phage is then incubated with bacteria at 37 ° C. for 30 minutes, so that the phage is used to produce 10 ml of logarithmically growing E. coli. Infect E. coli TG1. Then E. E. coli are plated on TYE plates containing 1% glucose and 100 μg / ml ampicillin. The resulting bacterial library is then
Rescue with Δgene 3 helper phage as described above to prepare phage for the next round of selection. The process was then repeated for all four affinity purifications, with tube washes with PBS, 0.1% Tw for the third and fourth time.
Increase 20 times with een-20 and 20 times with PBS.

Characterization of the binder. Using phage eluted from the third and fourth rounds of selection, E. coli HB 2151 is infected and soluble scFv is generated from a single colony for the assay (Marks et al., 1991). ELISA is performed using microtiter plates coated with either 10 pg / ml of a polypeptide of the invention in 50 mM bicarbonate, pH 9.6. ELISA
PCR fingerprinting (e.g. PCT publication WO
92/01047) and then further characterized by sequencing. These ELISA positive clones are also known in the art (eg, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody / antigen binding, and competitive agonist activity). Or competitive antagonist activity, etc.).

Example 32: Assay to detect stimulation or inhibition of B cell proliferation and differentiation
The generation of a functional humoral immune response is between B-lineage cells and their microenvironment,
Requires both soluble and cognate signaling. The signal can convey a positive stimulus (which causes the B-lineage cells to sustain programmed development) or a negative stimulus (which directs the cells to block the current generation pathway). To date, IL-2, IL-4, IL-5, IL-6, IL-7, IL
A number of stimulatory and inhibitory signals have been found to affect B cell responsiveness, including -10, IL-13, IL-14 and IL-15. Interestingly, these signals are weak effectors themselves, but can be combined with various costimulatory proteins to induce activation, proliferation, differentiation, homing, resistance, and death in the B cell population.

One of the most studied classes of B cell costimulatory proteins is the TNF superfamily. Within this family, CD40, CD27 and CD30 are
It has been found to modulate various immune responses with its respective ligands, CD154, CD70 and CD153. Assays that allow the detection and / or observation of the proliferation and differentiation of these B cell populations and their progenitor cells have the effect that various proteins can have on these B cell populations in terms of proliferation and differentiation. A valuable tool in making decisions. Listed below are two assays designed to allow detection of differentiation, proliferation, or inhibition of B cell populations and their precursors.

In Vitro Assay-Purified polypeptides of the invention, or truncated forms thereof, are evaluated for the ability to induce activation, proliferation, differentiation or inhibition and / or death in B cell populations and their precursors. . The activity of the polypeptides of the present invention on purified human tonsil B cells (measured qualitatively over a dose range of 0.1-10,000 ng / mL) is assessed in a standard B lymphocyte costimulation assay. . In this assay, purified tonsil B cells are cultured in the presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or fixed anti-human IgM antibody as a priming factor. Second signal, such as IL-2 and IL-15, as measured by tritiated thymidine incorporation, induces B cell proliferation cooperativity with SAC and IgM crosslinking. New cofactors can be readily identified using this assay. This assay involves isolating human tonsil B cells by magnetic bead (MACS) depletion of CD3 positive working cells. The resulting cell population, when evaluated by the expression of the CD45R (B220), it is often B cell than 95%.

Each sample of various dilutions was placed in an individual well of a 96-well plate, where culture medium (10% FBS, 5 × 10 ⁻⁵ M 2ME, 100 U / ml
Add 10 ⁵ B cells in a total volume of 150 μl suspended in RPMI 1640) containing penicillin, 10 μg / ml streptomycin, and 10 ⁻⁵ dilution of SAC. Growth or inhibition is started 72 hours after the addition of the factor, and 3H-
Quantify with thymidine (6.7 Ci / mM) with 20 h pulses (1 μCi / well). The positive and negative controls are IL2 and medium, respectively.

In Vivo Assay-BALB / c mice are injected twice daily (intraperitoneally) with buffer alone or 2 mg / kg of the polypeptide of the invention, or a truncated form thereof. Mice were given this treatment for 4 consecutive days at which time they were sacrificed and various tissues and sera were collected for analysis. Comparison of H & E sections from normal spleen and spleen treated with the polypeptides of the invention results in the activity of this polypeptide in spleen cells, e.g. periarterial lymphatic sheath spread and / or red pulp region A significant increase in nucleated cellularity, which can indicate activation of B cell population differentiation and proliferation, is confirmed. Anti-CD, a B cell marker
Using immunohistochemical studies with 45R (B220), a vaguely defined B cell compartment in which any physiological change to spleen cells (eg, spleen tissue disruption) infiltrates established T cell regions To determine whether it is due to an increase in B cell presentation.

Using flow cytometric analysis of spleens from mice treated with the polypeptide, the polypeptide is ThB + CD45R (B220) dull B
It shows whether the ratio of cells specifically increases over that observed in control mice.

Similarly, the presumed result of in vivo presentation of increased mature B cells is a relative increase in serum Ig titer. Therefore, serum IgM levels and serum IgA levels are compared between buffer treated and polypeptide treated mice.

The tests described in this example tested the activity of the polypeptides of the present invention.
However, those skilled in the art will recognize the activity (eg, gene therapy) of the polynucleotides of the invention,
The exemplified studies can be readily modified to test agonists and / or antagonists of the polynucleotides or polypeptides of the invention.

Example 33: T cell proliferation assay
(Proliferation assay for resting PBL)
CD3-induced proliferation assays are performed with PBMC and measured by ³ H-thymidine incorporation. This assay is performed as follows. A 96-well plate was prepared using 100 mAb of CD3 (HIT3a, Pharmingen)
μl / well, or isotype matched control mAb (B33.1) (
0.05 M bicarbonate buffer, pH 9.5, 1 μg / ml) overnight at 4 ° C.
Coat and then wash 3 times with PBS. PBMC are isolated from human peripheral blood by F / H gradient centrifugation and various concentrations of TNFδ and / or TNF
in RPMI containing 10% FCS and P / S in the presence of ε protein, m
Add to ⁴ wells (5 × 10 ⁴ / well) of Ab coated plate (total volume 200 μl). The relevant protein buffer and medium alone are controls. After 48 hours of incubation at 37 ° C., the plate is spun at 1000 rpm for 2 minutes and 100 μl of the supernatant is removed, and if an effect on proliferation is observed, IL-2 (or other cytokine) Stored at −20 ° C. for measurement.
The wells are supplemented with 100 μl medium containing 0.5 μCi ³ H-thymidine,
And it culture | cultivates at 37 degreeC for 18-24 hours. Wells were collected and ³ H-thymidine incorporation was used as an indicator of proliferation. Anti-CD3 alone is a positive control for proliferation. IL-2 (100 U / ml) is also used as a control to enhance proliferation. Control antibodies that do not induce T cell proliferation are TNFδ and / or
Or as a negative control for the effect of TNFε protein.

Alternatively, proliferation assay on PBL (peripheral blood lymphocytes) are resting, ³
Measured by H-thymidine incorporation. This assay is performed as follows. PBMC from human peripheral blood, Ficoll (LSM, ICN Biotech
nologies, Aurora, Ohio) isolated by gradient centrifugation and 10% (Fetal Calf Serum, Biofluids, Rock)
(ville, MD) / RPMI (Gibco BRL, Gaithersber)
g, MD) overnight in culture. With this overnight incubation period,
Adherent cells adhere to plastic. This leads to a lower background in the assay. This is because there are few cells that can act as antigen-presenting cells or produce growth factors. The next day non-adherent cells are collected, washed and used in proliferation assays. This assay is performed in 96 well plates with 2 × 10 ⁴ cells / well in a final volume of 200 μl. Supernatants expressing the protein of interest (eg, CHO or 293T supernatant) are tested at a final dilution of 30%, thus 60 μl is replaced with 140 μl of 10% F containing cells.
Add to CS / RPMI. Control supernatants are used at the same final dilution and express the following proteins: vector (negative control), IL-2 ( ^* ),
IFNγ, TNFα, IL-10 and TR2. In addition to control supernatant, recombinant human IL-2 (R & D Systems, Minneapolis, MN)
Is also used at a final concentration of 100 ng / ml. After 24 hours of culture, 1 μl of each well
Pulse with Ci ³ H thymidine (Nen, Boston, Mass.). Cells are then harvested 20 hours after the pulse and ³ H thymidine incorporation is used as an indicator of proliferation. Results are expressed as the mean ± standard error of triplicate samples.
( ^* ) Control cytokine I produced in each transfection
The amounts of L-2, IFNγ, TNFα and IL-10 are 300 pg to 5 ng / m.
vary between l.

(Co-stimulation assay)
Co-stimulation assays with resting PBL (peripheral blood lymphocytes) are performed in the presence of antibodies immobilized against CD3 and CD28. The use of antibodies specific for the constant region of CD3 mimics the induction of T cell activity that occurs through stimulation of T cell receptors by antigens. TC in the absence of costimulatory signal (second signal)
Cross-linking of R (first signal) results in a very low induction of proliferation and ultimately results in an “anergy” state (this condition is characterized by lack of proliferation and inability to produce cytokines). Arise. Addition of a costimulatory signal, such as an antibody to CD28, that mimics the action of a costimulatory molecule. B7-1 expressed on activated APC results in enhanced T cell responses including cell survival and IL-2 production. Therefore, this type of assay can detect both positive and negative effects on T cell proliferation caused by the addition of a supernatant expressing the protein of interest.

This assay is performed as follows. 100 ng / m with a final volume of 100 μl
l Anti-CD3 and 5 μg / ml anti-CD28 (Pharmingen, San D
coat 96 well plates and incubate overnight at 4 ° C. Wash plate twice with PBS before use. PBMCs from human peripheral blood, Ficoll (LSM, ICN Biotechnology)
s, Aurora, Ohio) isolated by gradient centrifugation and 10% FCS
(Fetal Calf Serum, Biofluids, Rockville
e, MD) / RPMI (Gibco BRL, Gaithersburg, MD)
) Incubate overnight. This overnight incubation period allows adherent cells to adhere to the plastic. This leads to a lower background in the assay. This is because there are few cells that can act as antigen-presenting cells or produce growth factors. The next day non-adherent cells are collected, washed and used in proliferation assays. This assay is 2 × 1 in a final volume of 200 μl.
Performed in 96-well plates using the 0 ⁴ cells / well. Supernatants expressing the protein of interest (eg, CHO or 293T supernatant) are tested at a final dilution of 30%, thus 60 μl is replaced with 140 μl of 10% FCS / R containing cells.
Add to PMI. Control supernatants are used at the same final dilution and express the following proteins: vector only (negative control), IL-2, IFNγ,
TNFα, IL-10 and TR2. In addition to the control supernatant, recombinant human I
L-2 (R & D Systems, Minneapolis, MN) is also used at a final concentration of 10 ng / ml. After 24 hours of culture, each well is pulsed with 1 μCi of ³ H thymidine (Nen, Boston, Mass.). The cells are then pulsed 2
Harvested after 0 hours and ³ H thymidine incorporation is used as an indicator of proliferation.
Results are expressed as the mean ± standard error of triplicate samples.

(Co-stimulation assay: IFNγ and IL-2 ELISA)
This assay is performed as follows. 300 ng / m with a final volume of 500 μl
Either l or 600 ng / ml anti-CD3 and 5 μg / ml anti-CD2
Coat 24-well plates with 8 (Pharmingen, San Diego, Calif.) And incubate overnight at 4 ° C. Wash plate twice with PBS before use. PBMCs from human peripheral blood, Ficoll (LSM,
ICN Biotechnologies, Aurora, Ohio) isolated by gradient centrifugation and 10% FCS (Fetal Calf Serum,
Biofluids, Rockville, MD) / RPMI (Gibco B
RL, Gaithersburg, MD) overnight. This overnight incubation period allows adherent cells to adhere to the plastic. This leads to a lower background in the assay. This is because there are few cells that can act as antigen-presenting cells or produce growth factors. The next day non-adherent cells are collected, washed and used in a costimulation assay. The assay is performed in 24-well plates pre-coated with 1 × 10 ⁵ cells / well in a final volume of 900 μl. Supernatant expressing the protein of interest (293T supernatant) is tested at a final dilution of 30%, so 300 μl is added to 600 μl of 10% FCS / RPMI containing cells. Control supernatants are used at the same final dilution and express the following proteins: vector only (
Negative control), IL-2, IFNγ, IL-12 and IL-18. In addition to the control supernatant, recombinant human IL-2 (all cytokines are R & D
(Purchased from Systems, Minneapolis, MN) is also used at a final concentration of 10 ng / ml, IL-12 at a final concentration of 1 ng / ml and IL-18 at a final concentration of 50 ng / ml. Control and unknown samples are tested in duplicate. Supernatant samples (250 μl) are collected on days 2 and 5 after the start of the assay. RISA for testing for secretion of IFNγ and IL-2
& D Systems using a kit purchased from Minneapolis, Minn. Results are expressed as the mean ± standard error of duplicate samples.

(Proliferation assay of preactivated resting T cells)
Proliferation assays with preactivated resting T cells are performed on cells that have been previously activated with lectin phytohemaglutinin (PHA). Lectins are polymeric plant proteins that can bind to residues on T cell surface glycoproteins, including TCRs, and act as polyclonal activators. PBL
Are treated with PHA and then cultured in the presence of low doses of IL-2 like effector T cells. These cells are generally more sensitive to further activation induced by growth factors such as IL-2. This is due to the expression of the high affinity IL-2 receptor. This receptor makes this population responsive to the amount of IL-2 (one hundredth of the effect it has on naive T cells). Thus, the use of this type of cell can detect the effects of very low doses of unknown growth factors that are not sufficient to induce proliferation on resting (naïve) T cells.

This assay is performed as follows. PBMC are isolated from human peripheral blood by F / H gradient centrifugation and cultured in 10% FCS / RPMI for 3 days in the presence of 2 μg / ml PHA (Sigma, Saint Louis, MO). The cells were then washed in PBS and in the presence of 5 ng / ml human recombinant IL-2 (R & D Systems, Minneapolis, Minn.) 10% FCS (Fetal Calf Serum, Biofluids, Rockville, MD) / RPMI Incubate in (Gibco BRL, Gaithersburg, MD) for 3 days. The cells were washed and rested in starvation medium (1% FCS / RPMI) for 16 hours before the proliferation assay was started. An aliquot of cells is analyzed by FACS to determine the percentage of T cells (CD3 positive cells) present; this usually ranges between 93-97% depending on the donor. This assay is performed in 96 well plates with 2 × 10 ⁴ cells / well in a final volume of 200 μl. Supernatants expressing the protein of interest (eg, CHO or 293T supernatant) are tested at a final dilution of 30%, therefore 60 μl is added to 140 μl of 10% FCS / RPMI containing cells. Control supernatants are used at the same final dilution and express the following proteins: vector (negative control), IL-2, IFNγ, TNFα, IL-10 and TR2. In addition to the control supernatant, recombinant human IL-2 is also used at a final concentration of 10 ng / ml. After 24 hours of culture, each well is pulsed with 1 μCi of ³ H thymidine (Nen, Boston, Mass.). Cells are then harvested 20 hours after the pulse and ³ H thymidine incorporation is used as an indicator of proliferation. Results are expressed as the mean ± standard error of triplicate samples.

The studies described in this example tested the activity of the polypeptides of the invention. However, those skilled in the art will recognize the activity (eg, gene therapy) of the polynucleotides of the invention.
The exemplified studies can be readily modified to test agonists and / or antagonists of the polynucleotides or polypeptides of the invention.

Example 34: Expression of MHC class II, costimulatory and adhesion molecules, and effect of polypeptides of the invention on cell differentiation of monocytes and monocyte-derived human dendritic cells
Dendritic cells are generated by the proliferation of proliferative progenitors found in peripheral blood: adherent PBMC or cleaned monocyte fractions are obtained from GM-CSF (50 ng / ml) and I
Incubate with L-4 (20 ng / ml) for 7-10 days. These dendritic cells are characterized by the characteristic phenotypes of immature cells (CD1, CD80, CD86, CD40 and M
HC class II antigen expression). Treatment with an activator such as TNF-α causes rapid changes in the surface phenotype (increased expression of MHC classes I and II, costimulatory and adhesion molecules, down-regulation of FCγRII, up-regulation of CD83) Arise. These changes are associated with increased antigen presentation capacity and functional maturation of dendritic cells.

FACS analysis of the surface antigen is performed as follows. Cells are treated with increasing concentrations of the polypeptide of the invention or LPS (positive control) for 1-3 days and treated with 1% B
Wash with PBS containing SA and 0.02 mM sodium azide, then 1
Incubate with 20 dilutions of appropriate FITC-labeled or PE-labeled monoclonal antibody at 4 ° C. for 30 minutes. After further washing, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Effect on the production of cytokines Cytokines produced by dendritic cells, particularly IL-12, are important in the initiation of T cell-dependent immune responses. IL-12
Strongly influences the development of Th1 helper T cell immune responses and cytotoxic T
Induces cell function and NK cell function. Using ELISA, IL as follows
-12 release is measured. Dendritic cells (10 ⁶ / ml) are treated with increasing concentrations of the polypeptides of the invention for 24 hours. LPS (100 ng / ml) is added to the cell culture as a positive control. The supernatant from the cell culture is then collected and a commercially available ELISA kit (eg R & D Systems (Minneap
olis, MN)) for IL-12 content. Use the standard protocol provided in the kit.

Effect on the expression of MHC class II, costimulatory molecules and adhesion molecules. Three major families of cell surface antigens can be identified on monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptors. Modulation of the expression of MHC class II antigens and other costimulatory molecules (eg, B7 and ICAM-1) can cause changes in monocyte antigen-presenting ability and ability to induce T cell activation. Increased Fc receptor expression may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.

FACS analysis is used to test surface antigens as follows. Monocytes are enriched with increasing concentrations of the polypeptide of the invention or LPS (positive control).
PB treated for ~ 5 days and containing 1% BSA and 0.02 mM sodium azide
Wash with S, then incubate with appropriate FITC-labeled or PE-labeled monoclonal antibody at 1:20 dilution for 30 minutes at 4 ° C.
After further washing, the labeled cells were washed with FACScan (Becton Dickin
son) flow cytometry.

Assays for molecules that activate (or inactivate) monocytes and / or increase monocyte survival (or decrease monocyte survival) (increasing monocyte activation and / or survival) Known in the art, and can be routinely applied to determine whether the molecules of the invention function as monocyte inhibitors or activators. The polypeptides, agonists or antagonists of the invention can be screened using the three assays described below. For each of these assays, peripheral blood mononuclear cells (PBMC) were expressed as Histop.
Single donor leuko by centrifugation through an aque gradient (Sigma)
pack (American Red Cross, Baltimore, MD
). Monocytes are countercurrent centrifugal elutriation (counterflo)
w centrifugal elimination) from PBMC.

Monocyte Survival Assay Human peripheral blood monocytes gradually lose viability when cultured in the absence of serum or other stimuli. Their death arises from an internally regulated process (apoptosis). The addition of an activator, such as TNFα, to the culture dramatically improves cell survival and prevents DNA fragmentation. Propidium iodine (
PI) Staining is used to measure apoptosis as follows. Monocytes, 100n
Incubate for 48 hours in serum-free medium (positive control) in polypropylene tubes in the presence of g / ml TNF-α (negative control) and in the presence of various concentrations of compound to be tested. Cells are suspended at a final concentration of 5 μg / ml in PBS containing PI to a concentration of 2 × 10 ⁶ / ml and then incubated for 5 minutes at room temperature before FACScan analysis. PI uptake has been shown to correlate with DNA fragmentation in this test paradigm.

Impact on cytokine release An important function of monocytes / macrophages is their regulatory activity on other cell populations of the immune system through the release of cytokines after stimulation. An ELISA for measuring cytokine release is performed as follows. Human monocytes are incubated at a density of 5 × 10 ⁵ cells / ml with increasing concentrations of the polypeptide of the invention and under the same conditions but in the absence of this polypeptide. For IL-12 production, the cells are primed overnight with IFN (100 U / ml) in the presence of a polypeptide of the invention. LPS (10 ng / ml) is then added. Conditioned medium is collected after 24 hours and stored frozen until use. TNF-α, IL-10, MCP-1 and IL-8 are then measured using a commercially available ELISA kit (eg, R & D Systems Minneapolis, MN) and applying the standard protocol provided with the kit To do.

(Oxidative burst) 96 purified monocytes
Plate well plate at 2-1 × 10 ⁵ cells / well. Increasing concentrations of the polypeptide of the present invention were added to a culture medium (RPMI 1640+
10% FCS, glutamine and antibiotics). After 3 days of incubation, the plate is centrifuged and the medium is removed from the wells. To a monolayer of macrophages, 0.2 ml of phenol red solution (140 m
M NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U / ml HRPO)
Add with stimulant (200 nM PMA). This plate is heated at 37 ° C for 2
Incubate for time and stop the reaction by adding 20 μl 1N NaOH per well. Read absorbance at 610 nm. In order to calculate the amount of H ₂ O ₂ produced by macrophages, a standard curve of a known molar concentration of H ₂ O ₂ solution is performed for each experiment.

The studies described in this example tested the activity of the polypeptides of the invention. However, those skilled in the art will recognize the activity of the polypeptide of the present invention, the activity of the polynucleotide (
The exemplified studies can be readily modified to test, for example, gene therapy), agonist activity, and / or antagonist activity.

(Example 35: Biological effect of the polypeptide of the present invention)
(Astrocyte and neural assay)
As described above, the recombinant polypeptide of the present invention expressed and purified in Escherichia coli is used for the activity of promoting cortical neuron cell survival, neurite growth or phenotypic differentiation, and glial fibrillary acidic protein immunity. Positive cells, astrocytes can be tested for induction of proliferation. The selection of cortical cells for bioassays is based on the prevailing expression of FGF-1 and FGF-2 in the cortical structure, as well as previously reported enhancement of cortical neuron survival resulting from FGF-2 treatment. A thymidine incorporation assay can be used, for example, to elucidate the activity of a polypeptide of the invention on these cells.

In addition, FGF to cortical or hippocampal neurons in vitro.
-2 (basic FGF) previous reports have demonstrated an increase in both neuronal survival and neurite outgrowth (Wallike et al., "Fibroblast growth factor promoters".
survival of dissociated hippocampal
neurons and enhances neurite extensi
on "Proc. Natl. Acad. Sci. USA 83: 3012-30
16 (1986), the assay is incorporated herein by reference in its entirety)
. However, reports from experiments performed on PC-12 cells indicate that these two responses are not necessarily synonymous and which FGFs are being tested as well as which receptors are being expressed in the target cells. It suggests that it can depend. The ability of the polypeptides of the invention to induce neurite outgrowth can be compared to the response obtained with FGF-2 using a primary cortical neuron culture paradigm, eg, using a thymidine incorporation assay.

(Fibroblast and endothelial cell assay)
Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.) And maintained in growth medium from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, Calif.). For proliferation assays, human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells / well for 1 day in 96-well plate growth medium. The cells are then incubated for 1 day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test protein for 3 days. Alamar Blue (Almar Bios
(Ciences, Sacramento, CA) is added to each well to a final concentration of 10%. The cells are incubated for 4 hours. Cell viability as C
Measure by reading with a ytoFluor fluorescent reader. For the PGE ₂ assay, human lung fibroblasts are cultured at 5,000 cells / well for 1 day in 96 well plates. After converting the medium to 0.1% BSA basal medium, the cells were
Incubate with FGF-2 or a polypeptide of the invention for 24 hours with or without L-1α. The supernatant is collected and the EIA kit (
Assay for PGE ₂ by Cayman, Ann Arbor, MI). For the IL-6 assay, human lung fibroblasts are cultured at 5,000 cells / well for 1 day in 96 well plates. After conversion of the medium to 0.1% BSA basal medium, the cells are incubated with FGF-2 for 24 hours with or without the polypeptide IL-1α of the present invention. The supernatant is collected and IL is obtained by ELISA kit (Endogen, Cambridge, Mass.)
Assay for -6.

Human lung fibroblasts are cultured with FGF-2 or a polypeptide of the invention in basal medium for 3 days, after which Alam is used to assess the effect on fibroblast proliferation.
Add ar Blue. FGF-2 should exhibit a stimulation of 10-2500 ng / ml that can be used comparable to stimulation with a polypeptide of the invention.

(Parkinson model)
The loss of motor function in Parkinson's disease is due to striatal dopamine deficiency resulting from degeneration of nigrostriatal dopaminergic projection neurons. A widely characterized animal model of Parkinson's disease involves systemic administration of 1-methyl-4phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken up by astrocytes and catabolized and released by monoamine oxidase B to 1-methyl-4-phenylpyridine (MPP ⁺ ). Subsequently, MPP ⁺ actively accumulates in dopaminergic neurons through dopamine's high affinity reuptake transporter. MPP ⁺ is then concentrated in the mitochondria by an electrochemical gradient and selectively inhibits nicotinamide adenine diphosphate: ubiquinone oxidoreductase (complex I), thereby preventing electron transfer, and Finally, active oxygen is generated.

It has been demonstrated in a tissue culture paradigm that FGF-2 (basic FGF) has trophic activity on nigral dopaminergic neurons (Ferrari)
Dev. Biol. 1989). Recently, Dr. Unsikker's group has demonstrated that FGF-2 administration with striatal gel-type implants results in almost complete protection of nigrodopaminergic neurons from toxicity associated with MPTP exposure ( Otto and Unsicker, J. Neuroscience,
1990).

Based on the data using FGF-2, the polypeptide of the present invention shows whether the polypeptide of the present invention has an action similar to that of FGF-2 in enhancing survival of dopaminergic neurons in vitro. And the polypeptides of the present invention can also test dopaminergic neurons in the striatum in vivo for protection from damage associated with MPTP treatment. The potential effects of the polypeptides of the invention are first tested in vitro in a dopaminergic neuronal cell culture paradigm. Wis on the 14th day of pregnancy
Cultures are prepared by dissecting the midbrain floor plate from tar rat embryos. Tissues were separated with trypsin and plated at a density of 200,000 cells / cm ^{2 on} coverslips coated with polyortinin-laminin. The cells are maintained in F12 medium containing Dulbecco's modified Eagle medium and hormone supplement (N 1 ). After 8 days in vitro, cultures are fixed with paraformamide and processed for immunohistochemical staining with tyrosine hydroxylase (a specific marker for dopaminergic neurons). Separate cell cultures are prepared from embryonic rats. The culture medium is changed every 3 days and this factor is also added at each time point.

Since dopaminergic neurons are isolated from animals at 14 days of gestation (this time of development is past the stage where dopaminergic progenitor cells proliferate), an increase in the number of tyrosine hydroxylase immunopositive neurons survives in vitro. Shows an increase in the number of dopaminergic neurons. Thus, if the polypeptide of the present invention acts to prolong dopaminergic neuron survival, this suggests that the polypeptide may be involved in Parkinson's disease.

The studies described in this example tested the activity of the polypeptides of the invention. However, those skilled in the art will recognize the activity (eg, gene therapy) of the polynucleotides of the invention.
The exemplified studies can be readily modified to test the activity of the agonists and / or antagonists of the present invention.

Example 36: Effect of the polypeptide of the present invention on the proliferation of vascular endothelial cells
On day 1, human umbilical vein endothelial cells (HUVEC) were collected from 4% fetal bovine serum (FBS).
), 16 units / ml heparin, and M199 containing 50 units / ml endothelial cell growth supplement (ECGS, Biotechnique, Inc.).
Seed in medium at a density of 2-5 × 10 ⁴ cells per 35 mm dish. On day 2, the medium was M199 containing 10% FBS, 8 units / ml heparin.
Replace with. A polypeptide having the amino acid sequence of SEQ ID NO: Y and a positive control (eg, VEGF and basic FGF (bFGF)) are added at various concentrations. On day 4 and 6, change medium. Day 8, Coulter C
Determine the cell number using the counter.

An increase in the number of HUVEC cells indicates that the polypeptide of the present invention can proliferate vascular endothelial cells.

The studies described in this example tested the activity of the polypeptides of the invention. However, those skilled in the art will recognize the activity (eg, gene therapy) of the polynucleotides of the invention.
The exemplified studies can be readily modified to test the activity of the agonists and / or antagonists of the present invention.

(Example 37: Stimulating effect of the polypeptide of the present invention on the proliferation of vascular endothelial cells)
Colorimetric MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxyl) using the electron coupling reagent PMS (phenazine methosulfate) for the evaluation of growth factor mitogenic activity (Methoxyphenyl) -2- (4-sulfophenyl) 2H-tetrazolium) assay was performed (CellTiter
96 AQ, Promega). Cells are seeded in 96-well plates (5,000 cells / well) in 0.1 mL of serum supplemented media and allowed to attach overnight. 0
. After serum starvation in 5% FBS for 12 hours, conditions (polypeptides of the invention in bFGF, VEGF ₁₆₅ or 0.5% FBS) with or without Heparin (8 U / ml) for 48 hours Added. 20 mg MTS / PMS
The mixture (1: 0.05) is added per well and allowed to incubate for 1 hour at 37 ° C., after which the absorbance at 490 nm is measured with an ELISA plate reader. The background absorbance of control wells (with medium, without cells) is subtracted and 7 wells are run in parallel for each condition. Leak et al., In Vitro Cell. Dev. Biol. 30A: 5
12-518 (1994).

The studies described in this example tested the activity of the polypeptides of the invention. However, those skilled in the art will recognize the activity (eg, gene therapy) of the polynucleotides of the invention.
The exemplified studies can be readily modified to test the activity of the agonists and / or antagonists of the present invention.

(Example 38: Inhibition of PDGF-induced vascular smooth muscle cell proliferation stimulating effect)
HAoSMC proliferation can be measured, for example, by BrdUrd incorporation. Briefly, subconfluent quiescent cells growing on 4-chamber slides are labeled as C
Transfect with RP or FITC labeled AT2-3LP. The cells are then pulsed with 10% bovine serum and 6 mg / ml BrdUrd. 24
After time, immunocytochemistry is performed by using a BrdUrd staining kit (Zymed Laboratories). Briefly, the cells are incubated with a biotinylated mouse anti-BrdUrd antibody for 2 hours at 4 ° C. after exposure to denaturing solution, followed by incubation with streptavidin-peroxidase and diaminobenzidine. After counterstaining with hematoxylin, the cells are sampled for microscopic examination and Brd Urd-positive cells are counted. Brd
The Urd coefficient is calculated as the ratio of BrdUrd-positive cells to the total number of cells. In addition, simultaneous detection of BrdUrd staining (nucleus) and FITC uptake (cytoplasm) is performed on individual cells by simultaneous use of both bright field illumination and dark field UV fluorescent illumination. (Hayashida et al., J. Biol. Chem. 6: 271 (
36): 21985-21992 (1996)).

The studies described in this example tested the activity of the polypeptides of the invention.
However, those skilled in the art will recognize the activity (eg, gene therapy) of the polynucleotides of the invention,
In order to test the activity of the agonists and / or antagonists of the present invention,
The illustrated study can be easily modified.

(Example 39: Stimulation of endothelial migration)
This example can be used to explore the possibility that the polypeptides of the present invention may stimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48-well microchemotaxis chamber (Neuroprobe Inc., Cabin John, MD;
W et al. Immunological Methods 1980; 33:
239-247). Polycarbonate filter free of polyvinylpyrrolidone (with 8 μm pore size) (Nucleopore Corp. Cam
bridge, MA) at room temperature for at least 6 hours with 0.1% gelatin,
It is then dried under sterile air. Test substances are diluted to the appropriate concentration in M199 supplemented with 0.25% bovine serum albumin (BSA) and a final dilution of 25 μl is placed in the bottom chamber of the modified Boyden device. Subconfluent, early passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for the minimum time required for cell detachment. After placing the filter between the bottom and top chambers, seed 2.5 × 10 ⁵ cells suspended in 50 μl M199 containing 1% FBS in the upper compartment. The device is then
Cells were allowed to migrate by incubation for 5 hours at 37 ° C. in a chamber moistened with 5% CO 2. After the incubation period, the filter is removed and the upper side of the filter with non-migrating cells is placed on a rubbery policeman (polic
eman). The filter is fixed with methanol and Giem
Stain with sa solution (Diff-Quick, Baxter, McGraw P
arc, IL). Migration is quantified by counting cells in 3 random high-power fields (40 ×) in each well. All groups are executed in quadruplicate.

The studies described in this example tested the activity of the polypeptides of the invention.
However, those skilled in the art will recognize the activity (eg, gene therapy) of the polynucleotides of the invention,
In order to test the activity of the agonists and / or antagonists of the present invention,
The illustrated study can be easily modified.

(Example 40: Stimulation of nitric oxide production by endothelial cells)
Release of nitric oxide by the vascular endothelium is thought to be a mediator of vascular endothelial relaxation. Thus, the activity of a polypeptide of the invention can be assayed by measuring nitric oxide production by endothelial cells in response to the polypeptide.

Nitric oxide was used for 24 hours of starvation followed by various levels of positive control (
For example, in a 96-well plate of confluent microvascular endothelial cells after 4 hours exposure to VEGF-1) and a polypeptide of the invention. Nitric oxide in the medium is quantified by use of Griess reagent to determine the total amount of nitrous acid after reduction of nitrate derived from nitric oxide by nitrate reductase. The effect of the polypeptides of the invention on nitric oxide release is tested in HUVEC.

Briefly, NO release from cultured HUVEC monolayers is measured by NO meter (Iso
-NO, World Precision Instruments Inc.
) Measurement is performed using a NO-specific polarographic electrode connected to (1049).
Calibration of the NO element is performed according to the following formula:
2KNO ₂ + 2KI + 2H ₂ SO ₄ 62NO + I ₂ + 2H ₂ O + 2K ₂ SO ₄
A calibration curve was obtained in graded concentrations of KNO ₂ (in a calibration solution containing KI and H ₂ SO _4.
0, 5, 10, 25, 50, 100, 250, and 500 nmol / L). The specificity of the Iso-NO electrode for NO is determined in advance by measuring NO from authentic NO gas (105
0). The culture medium is removed and the HUVECs are washed twice with Dulbecco phosphate buffered saline. Cells were then transferred to 5m in 6 well plates.
In order to maintain the temperature at 37 ° C., the slide warmer (Lab Lin)
e Instruments Inc. ). A NO sensor probe is inserted vertically into the well, and the tip of the electrode is held 2 mm below the surface of the solution before adding different conditions. S-nitrosoacetylpenicillamine (SNAP) is used as a positive control. The amount of NO released is expressed as picomolar concentration per 1 × 10 ⁶ endothelial cells. All reported values are an average of 4-6 measurements in each group (number of cell culture wells). Leak et al., Biochem. and B
iophys. Res. Comm. 217: 96-105 (1995).

The studies described in this example tested the polypeptide activity of the present invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonist, and / or antagonist activity of the polynucleotides of the invention.

(Example 41: Effect of the polypeptide of the present invention on cord formation in angiogenesis)
Another process in angiogenesis is a cord characterized by endothelial cell differentiation (c
ord) formation. This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) was used as a proliferating cell (passage 2)
Applications , Inc. Buy from Cell Applicat
Culture in ions' CADMEC Growth Medium and use at passage 5. For in vitro angiogenesis assays, the wells of a 48-well cell culture plate were transferred to Cell Applications' Attachment.
Using Factor Medium (200 ml / well) at 37 ° C. 3
Coat for 0 minutes. CADMEC are seeded into wells coated at 7,500 cells / well and cultured overnight in Growth Medium. Then this Gr
owth Medium may be added to a control buffer or a polypeptide of the present invention (
300 mg Cell Applicati containing 0.1-100 ng / ml)
Replace with ons' Chord Formation Medium and incubate the cells for an additional 48 hours. The number and length of capillary-like cords can be determined using Boeckel
Quantify through use of er VIA-170 video image analyzer. All assays were performed in triplicate.

Commercially available (R & D) VEGF (50 ng / ml) is used as a positive control. b-estradiol (1 ng / ml) is used as a negative control. An appropriate buffer (no protein) is also used as a control.

The studies described in this example tested the activity of the polypeptides of the invention. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonist, and / or antagonist activity of the polynucleotides of the invention.

(Example 42: Angiogenic effect on chicken chorioallantoic membrane)
The chicken chorioallantoic membrane (CAM) is a well-established system for testing angiogenesis. Angiogenesis in the CAM can be easily visualized and quantified. The ability of the polypeptides of the invention to stimulate angiogenesis in the CAM can be tested.

Fertilized chicken White Leghorn (Gallus gallus) Contact and Japanese quail (qual) (Coturnix coturnix), incubated at 37.8 ° C. and 80% humidity. Differentiated CAM of quail embryos of chickens Li us and 13 days of age of the 16-day-old to study in the following way.

On the fourth day of development, a window is made in the eggshell of the chicken egg. The embryo is examined for normal development and the egg is sealed with cellotape®. 1 more
Incubate until day 3. Thermonox cover glass (Nunc,
Naperville, IL) is cut into discs about 5 mm in diameter. Dissolve sterile and salt-free growth factor in sterile water and pipet approximately 3.3 mg / 5 ml onto the disk. After air drying, the inverted disc is applied to the CAM. 3 days later, sample 3
Fix in% glutaraldehyde and 2% formaldehyde and 0.12
Rinse with M sodium cacodylate buffer. Stereomicroscope [Wild M8
] And embedded for semi-thin and ultrathin sectioning as described above. Control is performed using only the carrier disk.

The studies described in this example tested the polypeptide activity of the present invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonist, and / or antagonist activity of the polynucleotides of the invention.

Example 43: Angiogenesis assay using Matrigel grafts in mice
The in vivo angiogenesis assay of the polypeptides of the present invention measures the ability of existing capillary network-like structures to form new vessels in implanted capsules of mouse extracellular matrix material (Matrigel). This protein is liquid Ma at 4 ° C.
After mixing with trigel, the mixture is then injected subcutaneously into mice, where the mixture solidifies. After 7 days, Matrigel's solid “plug” is removed and tested for the presence of new blood vessels. Matrigel, Bect
on Dickinson Labware / Collaborative B
Purchase from iomedical Products.

The Matrigel material is a liquid when thawed at 4 ° C. This Matrige
l is mixed with the polypeptide of the invention at 150 ng / ml at 4 ° C. and withdrawn into a cold 3 ml syringe. Approximately 8 week old female C57B1 / 6 mice are injected with a mixture of Matrigel and experimental protein at two sites on the mid-ventral aspect of the abdomen (0
. 5 ml / site). Seven days later, the mice were sacrificed by cervical dislocation and Matrig
Remove and wash the el plug (ie, remove any adhering membrane and glandular tissue). Fix the same whole plug in neutral buffered 10% formamide,
Embed in paraffin and, after staining with Masson's Trichrome, use to make sections for histological examination. Process cross sections from three different regions of each plug. Selected sections are stained for the presence of vWF. The positive control for this assay is bovine basic FGF (150 n
g / ml). Matrigel alone is used to determine the basal level of angiogenesis.

The studies described in this example tested the polypeptide activity of the present invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonist, and / or antagonist activity of the polynucleotides of the invention.

(Example 44: Rescue of ischemia in rabbit lower limb model)
To study the in vivo effects of the polynucleotides and polypeptides of the invention on ischemia, a rabbit hindlimb ischemia model has been previously described (Takesh
ita et al., Am J. et al. Pathol 147: 1649-1660 (1995).
) By surgical removal of one femoral artery. Resection of the femoral artery results in retrograde propagation of the thrombus and occlusion of the external iliac artery. As a result, blood flow to the ischemic limb depends on collateral vessels originating from the internal iliac artery (Takeshita et al., Am J
. Pathol 147: 1649-1660 (1995)). At 10-day intervals, rabbits are allowed to recover after surgery and develop endogenous collateral vessels. Surgery (0
Day) On the 10th day after baseline angiography, the internal iliac artery of the ischemic limb was
Using 500 mg of naked expression plasmid containing the polynucleotide of the present invention, (
Riessen et al., Hum Gene Ther. 4: 749-758 (199
3); Leclerc et al. Clin. Invest. 90: 936-944
(1992)) and is transfected by arterial gene transfer techniques using hydrogel-coated balloon catheters. When the polypeptide of the invention is used for treatment, a single bolus of 500 mg of the polypeptide of the invention or control is delivered through the infusion catheter for 1 minute into the internal iliac artery of the ischemic limb. On day 30, various parameters are measured in these rabbits:
(A) BP ratio—ratio of ischemic limb systolic blood pressure to normal limb systolic blood pressure; (b) blood flow and reflux— resting FL: blood flow during non-diastolic state, and maximum FL: complete extended to
Blood flow (which is also known as an indirect indicator of vascular volume) between the state and reflux, the maximum F
L: reflected in the ratio of resting FL; (c) angiographic score (angiographic)
Score) —This is measured by angiography of the collateral vessels. Score is determined by the percentage of circles in the overlay grid (using transverse opacified arteries divided by the total number of rabbit thighs m); (d) capillary density—number of collateral capillaries Determined in light microscopy sections obtained from hind limbs.

The studies described in this example tested the polynucleotide and polypeptide activities of the present invention. However, one of ordinary skill in the art can readily modify the illustrated studies to test the agonists and / or antagonists of the present invention.

(Example 45: Effect of the polypeptide of the present invention on vasodilation)
Since dilation of the vascular endothelium is important for lowering blood pressure, spontaneously hypertensive rats (S
The ability of the polynucleotide of the invention to affect blood pressure in HR) is tested. Increasing doses (0, 10, 30, 100, 300, and 900 mg / kg) of the polypeptides of the invention are administered to 13-14 week old spontaneously hypertensive rats (SHR). Data are expressed as mean +/- SEM. Statistical analysis is performed using a two-tailed t-test and statistical significance is defined as p <0.05 vs response to buffer alone.

The studies described in this example tested the polypeptide activity of the present invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonist, and / or antagonist activity of the polynucleotides of the invention.

(Example 46: Rat ischemic skin flap model)
Evaluation parameters include skin blood flow, skin temperature, and factor VIII immunohistochemistry or endothelial alkaline phosphatase response. The expression of the polypeptide of the present invention during skin ischemia is studied using in situ hybridization.

The study in this model is divided into three parts:
a) ischemic skin b) ischemic skin wound c) normal wound.

The experimental protocol includes:
a) 3 × 4 cm single stem full thickness random skin flap
A full-thickness random skin flap (muscle flap over the lower back of the animal) is generated.

b) Create excisional wound in ischemic skin (skin flap) (diameter 4-6mm)
c) Topical treatment of excised wounds (days 0, 1, 2, 3, 4 after wounding) with the polypeptides of the invention in the following different dosage ranges: 1 mg to 100 mg
d) For histological, immunohistochemical, and in situ studies,
Wound tissue is collected on days 3, 5, 7, 10, 14, and 21 after wounding.

The studies described in this example tested the polypeptide activity of the present invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonist, and / or antagonist activity of the polynucleotides of the invention.

(Example 47: peripheral artery disease model)
Angiogenesis treatment using the polypeptides of the present invention is a novel therapeutic strategy for obtaining restoration of blood flow around ischemia in the case of peripheral arterial disease. The experimental protocol includes:
a) One unilateral femoral artery is ligated to create the hindlimb ischemic muscle, while the other unilateral hindlimb serves as a control.

b) Deliver the polypeptide of the invention in a dosage range of 20 mg to 500 mg intravenously and / or intramuscularly three times per week (probably more) for 2-3 weeks.

c) The ischemic muscle tissue is collected at 1, 2, and 3 weeks after ligation of the femoral artery for analysis of expression of the polypeptide of the invention and histology. A biopsy is also performed on normal muscles in the contralateral hind limb on the other side.

The studies described in this example tested the polypeptide activity of the present invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonist, and / or antagonist activity of the polynucleotides of the invention.

(Example 48: Ischemic myocardial disease model)
The polypeptides of the present invention is evaluated as a potent mitogen capable of reconstructing the <br/> new blood vessels resulting stimulate the development of collateral vessels, and after coronary artery occlusion. Changes in polypeptide expression are investigated in situ. The experimental protocol includes:
a) The heart is exposed via the left thoracotomy of the rat. Immediately, the left coronary artery
Occlude with a thin suture (6-0) and close the thorax.

b) Deliver the polypeptide of the invention in a dosage range of 20 mg to 500 mg intravenously and / or intramuscularly three times (perhaps more) for 2-4 weeks per week.

c) 30 days after surgery, the heart is removed for morphometry and in situ analysis and cross-sectioned
That turn into.

The studies described in this example tested the polypeptide activity of the present invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonist, and / or antagonist activity of the polynucleotides of the invention.

(Example 49: Rat corneal wound healing model)
This animal model shows the effect of the polypeptides of the invention on neovascularization. The experimental protocol includes:
a) Make a 1-1.5mm long incision from the center of the cornea to the stromal layer b) Insert a spatula under the lip of the incision facing the outer edge of the eye c) Make a pocket (its base is From the edge of the eye (form) is 1 to 1.5 mm)
d) A small pill containing 50 ng to 5 μg (ug) of the polypeptide of the invention is placed in the pocket e) Treatment with the polypeptide of the invention is also within the dosage range of 20 mg to 500 mg (daily treatment) For 5 days) may be applied topically to the corneal wound.

The studies described in this example tested the polypeptide activity of the present invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonist, and / or antagonist activity of the polynucleotides of the invention.

Example 50: Diabetic mouse and glucocorticoid disorder wound healing model
(A. Diabetes db + / db + mouse model)
To demonstrate that the polypeptides of the invention promote the healing process, a genetic diabetes mouse model of wound healing is used. The full-thickness wound healing model in db + / db + mice is a well-characterized, clinically relevant and reproducible model of impaired wound healing. Diabetic wound healing relies on granulation tissue formation and re-epithelialization rather than contraction (Gartner, MH et al., J. Surg. Res. 52: 389).
1992); Greenhalgh, D .; G. Et al., Am. J. et al. Pathol. 1
36: 1235 (1990)).

This diabetic animal has many of the characteristic characteristics observed in type II diabetes mellitus. Homozygous (db + / db +) mice have their normal heterozygous (
db + / + m) Obese compared to litters. Mutant diabetes (db + / db +)
Mice have a single autosomal recessive mutation on the fourth chromosome of (db +) (C
oleman et al., Proc. Natl. Acad. Sci. USA 77:28
3-293 (1982)). Animals exhibit bulimia, polyposis, and polyuria. Mutant diabetic mice (db + / db +) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Ma
ndel et al. Immunol. 120: 1375 (1978); Debra
y-Sachs, M.M. Et al., Clin. Exp. Immunol. 51 (1): 1
-7 (1983); Leiter et al., Am. J. et al. of Pathol. 114:
46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular injury,
Basement membrane thickening and glomerular filtration abnormalities have been described in these animals (N
orido, F.M. Et al., Exp. Neurol. 83 (2): 221-232 (1
984); Robertson et al., Diabetes 29 (1): 60-67.
(1980); Giacomelli et al., Lab Invest. 40 (4):
460-473 (1979); Coleman, D .; L. Diabetes 3
1 (Addendum): 1-6 (1982)). These homozygous diabetic mice develop hyperglycemia, which is resistant to insulin similar to human type II diabetes (Mandel et al., J. Immunol. 120: 1375-1377).
(1978)).

The characteristics observed in these animals indicate that healing in this model can be similar to that observed in human diabetes (Greenhalgh et al.,
Am. J. et al. of Pathol. 136: 1235-1246 (1990)).

Genetically diabetic female C57BL / KsJ (db + / db +) mice and their non-diabetic (db + / + m) heterozygous littermates were used in this study (J
ackson Laboratories). These animals are purchased at 6 weeks of age. The start of the study is 8 weeks old. Animals are raised individually and given food and water ad libitum. All manipulations are performed using aseptic manipulation. This experiment is called Human Ge
name Sciences, Inc. Institutional Ani
mal Care and Use Committee and the G
uidelines for the Care and Use of La
Follow the rules and guidelines of the laboratory Animals.

The wound protocol was adapted from previously reported methods (Tsuboi, R. and Rif
kin, D.C. B. J. et al. Exp. Med. 172: 245-251 (1990)
). Briefly, on the day of wounding, the animal was dissolved in deionized water A
Anesthetize with intraperitoneal injection of vertin (0.01 mg / mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol. The animal's dorsal area is shaved and the skin is washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze before wounding. The 8 mm full thickness wound was then
Fabricate using Keyes tissue punch. Immediately after wounding, the surrounding skin is gently stretched to remove the wound spread. The wound is left open during the experiment. Treatment application is topically received for 5 consecutive days from the date of wounding. Prior to treatment, the wound is gently washed with sterile saline and gauze sponge.

The wound is visually inspected and photographed at a fixed distance on the day of surgery and every two days thereafter. Wound closure is determined by daily measurements on days 1-5 and 8. Wound calibrated Jameson caliper (c
caliber) and measure horizontally and vertically. A wound is considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

The polypeptides of the invention are administered in the vehicle for 8 days using different dosage ranges (4 mg to 500 mg per wound per day). The vehicle control group was given 50 mL of vehicle solution.

Animals are euthanized on day 8 by intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound and surrounding skin are then collected for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) vehicle placebo control, 2) untreated group, and 3) treated group.

Wound closure is analyzed by measuring the area on the horizontal and vertical axes and obtaining the total area of the wound. Shrinkage is then assessed by establishing the difference between the area of the initial wound (day 0) and the area after treatment (day 8). This wound area on day 1 is 64 mm ² (size corresponding to the skin punch). The calculation is performed using the following formula:
[Open area on day 8]-[Open area on day 1] / [Open area on day 1]
The specimen is fixed in 10% buffered formalin and the paraffin embedded mass is cut perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H & E) staining is performed on bisected wound cross sections. Wound histology is used to assess whether the healing process and morphological appearance of the repaired skin has been altered by treatment with the polypeptides of the invention. This assessment includes verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, reepithelialization, and epidermal maturation (Green
nhalgh, D.M. G. Et al., Am. J. et al. Pathol. 136: 1235 (19
90)). A blinded observer (blinded) with a calibrated lens micrometer
observer).

Tissue sections are also immunohistochemically stained with polyclonal rabbit anti-human keratin antibodies using the ABC Elite detection system. Human skin is used as a positive tissue control while nonimmune IgG is used as a negative control.
Keratinocyte proliferation is determined by assessing the extent of wound re-epithelialization using a calibrated lens micrometer.

Proliferating cell nuclear antigen / cyclin (PCNA) in skin specimens, ABC El
This is demonstrated by using an anti-PCNA antibody (1:50) with an ite detection system. Human colon cancer can serve as a positive tissue control, and human brain tissue can be used as a negative tissue control. Each specimen includes a section with primary antibody shedding and replacement with non-immune mouse IgG. The order of these sections is based on the extent of growth on a scale of 0-8 (lower scale reflecting slight growth to higher side reflecting intense growth).

Experimental data is analyzed using a one-sided t-test. A p value of <0.05 is considered significant.

(B. Steroid disorder rat model)
Inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids an
d Wound healing: Anti-Infrastructure St
eroid Action: Basic and Clinical Aspe
cts. 280-302 (1989); Wahl et al., J. MoI. Immunol. 1
15: 476-481 (1975); Werb et al., J. Biol. Exp. Med. 147
: 1684-1694 (1978)). Glucocorticoids inhibit angiogenesis, vascular permeability (Ebert et al., An. Inter. Med. 37: 7
01-705 (1952)), fibroblast proliferation, and collagen synthesis (Bec).
k et al., Growth Factors. 5: 295-304 (1991); Ha
ynes et al. Clin. Invest. 61: 703-797 (1978)
) As well as causing a transient decrease in circulating monocytes (Ha
ynes et al. Clin. Invest. 61: 703-797 (1978)
Wahl, “Glucocorticoids and wound heel;
ling ": Antiinflammation Steroid Actio
n: Basic and Clinical Aspects, Academi
c Press, New York, 280-302 (1989)) to delay wound healing. Systemic administration of steroids for impaired wound healing is a well-established phenomenon in rats (Beck et al., Growth Fac
tors. 5: 295-304 (1991); Haynes et al., J. Biol. Clin
. Invest. 61: 703-797 (1978); Wahl, “Gluc.
ocorticoids and wound healing ": Antii
nflammatory Steroid Action: Basic and
Clinical Aspects, Academic Press, New
York, 280-302 (1989); Pierce et al., Proc. Na
tl. Acad. Sci. USA 86: 2229-2233 (1989)).

To demonstrate that the polypeptides of the present invention can promote the healing process, the effect of multiple topical applications of the polypeptide on a full-thickness cutaneous wound in rats, where healing is impaired by systemic administration of methylprednisolone, is demonstrated. evaluate.

Young adult male Sprague Dawley rats (weight 250-300 g)
) (Charles River Laboratories) is used for this experiment. The animals are purchased at 8 weeks of age. And the start of the study is 9 weeks old. The healing response of the rat is based on systemic administration of methylprednisolone (17 m
g / kg / rat, intramuscular). Animals are raised individually and given food and water ad libitum. All manipulations are performed using aseptic techniques. This study is called H
Institution of uman Genome Sciences, Inc
al Animal Care and Use Committee and
the Guidelines for the Care and Use
of Laboratory Animals rules and guidelines.

The wound protocol follows Section A above. On the day of wounding, the animals were ketamine (50
anesthetize with an intramuscular injection of mg / kg) and xylazine (5 mg / kg). The animal's dorsal area is shaved and the skin is washed with 70% ethanol and iodine solution. The surgical area is dried with sterile gauze before wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch. During the experiment, it left open the wounds scratch.
Application of the test substance is given topically once a day for 7 consecutive days starting from the day of wounding and then methylmethylprednisolone administration. Prior to treatment, the wound is gently washed with sterile saline and gauze sponge.

The wound is visually inspected and photographed at a fixed distance on the day of wounding and at the end of treatment. Wound closure is determined by daily measurements on days 1-5 and on day 8. The wound is measured horizontally and vertically using a calibrated Jameson caliper. A wound is considered healed when granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

The polypeptides of the invention are administered in the vehicle for 8 days using different dose ranges (4 mg to 500 mg per wound per day). The vehicle control group received 50 mL of vehicle solution.

Animals are euthanized on day 8 by intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound and surrounding skin are then collected for histology. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing .

Four groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) untreated group, 2) vehicle placebo control, 3) treated group.

Wound closure is analyzed by measuring the area on the vertical and horizontal axes and obtaining the total area of the wound. Closure is then assessed by establishing the difference between the initial wound area (day 0) and the post-treatment (day 8) area. This wound area on day 1 is 64 mm ² (size corresponding to the skin punch). The calculation is performed using the following formula:
[Open area on day 8]-[Open area on day 1] / [Open area on day 1]
The specimen is fixed in 10% buffered formalin and the paraffin-embedded mass is cut perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H & E) staining is performed on bisected wound cross sections. The histological examination of the wound makes it possible to assess whether the healing process and morphological appearance of the repaired skin has been improved by treatment with the polypeptides of the invention. A graduated lens micrometer is used by a blinded observer to determine the distance of the wound gap.

Experimental data is analyzed using a one-sided t-test. A p value of <0.05 is considered significant.

The studies described in this example tested the polypeptide activity of the present invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the present invention.

(Example 51: Lymphedema animal model)
The purpose of this experimental approach is to create an appropriate and consistent model of lymphedema to test the therapeutic effects of the polypeptides of the invention in lymphangiogenesis and re-establishment of lymphatic circulation in the rat hind limb . Efficacy is measured by swollen volume of the affected limb, quantification of the amount of lymphatic vessels, total plasma protein, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progression of edema lasts up to 3-4 weeks.

Prior to beginning surgery, blood samples are collected for protein concentration analysis.
Male rats (body weight approximately 350 g) are dosed with pentobarbital. The right limb is then shaved from the knee to the hip joint. The shaved area is wiped with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. After measuring the circumference and volume, mark the two measurement levels (0.5 cm above the heel, midpoint of the back of the foot) and then inject the dye into the foot. To the endothelium of both the right and left footpads,
Inject 0.05 ml of 1% Evan's Blue. Next, after injection of the dye into the foot, circumference measurement and volume measurement are performed.

Using the knee joint as a mark, the inguinal groin incision is performed in an annular shape so that the position of the femoral blood vessel can be confirmed. Using forceps and a hemostatic agent, the skin flap is dissected and separated. After confirming the position of the femoral blood vessel, the lymphatic vessel running along the side of the blood vessel and below the blood vessel is located. The main lymphatic vessels in this area are then electrocoagulated suture ligated.

Using a microscope, the dorsal muscles of the limb (semitenendinosis)
Make a smooth incision in the vicinity of the adductor muscle. The location of the popliteal lymph node is then confirmed. The two proximal and two distal lymph vessels and the distal blood supply of the popliteal node are then ligated with sutures. The popliteal lymph nodes and any associated adipose tissue are then removed by cutting the connective tissue.

Be careful to manage any mild bleeding that occurs during this procedure. After biting the lymph vessels, the skin flap is placed on the liquid skin (Vetbond) (AJ Buck)
Seal by using. Separate skin edges are sealed to the underlying muscle tissue leaving a gap of about 0.5 cm around the leg. The skin may also be anchored by stitching to the underlying muscle when needed.

In order to avoid infection, animals are housed individually with mesh (no flooring). Recovered animals are checked daily through the optimal edema peak. This peak is typically occurs in up to 5 to 7 days. The plateau edema peak is then observed. To assess the intensity of lymphedema, the circumference and volume of two designated locations on each limb are measured before manipulation and daily for 7 days. The effect of plasma proteins on lymphedema will be determined and whether protein analysis is a useful test boundary will also be investigated. The weight of both the control and edema limbs is evaluated at two locations. Analysis is performed in a blind manner.

Circumference measurement: Under short-term gas anesthesia to prevent movement of the limb, the circumference of the limb is measured using a cloth tape measure. Measurements are taken by two different persons at the talus and dorsal foot, and then these two readings are averaged. Readings are taken from both the control and edema limbs.

Volume measurement: On the day of surgery, animals are anesthetized with pentobarbital and tested prior to surgery. For daily volumetric measurements, animals are placed under short-term halothane anesthesia (rapid fixation and quick recovery), both legs are shaved, and the legs are marked equally with water resistant markers. The legs are first soaked in water, then soaked in the device to each marked level, and then Buxco edema software (Chen / Vict
or). Data is recorded by one person, while others immerse up to the marked area.

Blood-plasma protein measurement: Blood is removed prior to surgery, centrifuged, and serum separated, and then concluded for total protein and Ca2 + comparison.

Limb weight comparison: After removing blood, the animals are prepared for tissue collection.
The limb is amputated with quillitine, then both experimental and control legs are ligated and amputated and weighed. A second weighing is performed by removing the tibio-cacanal joint and weighing the foot.

Histological preparation: The transverse muscle located behind the knee (popliteal) region is cut out and placed in a metal mold, filled with freezeGel, soaked in cold methylbutane and placed at -80EC until cut into a labeled sample bag . During amputation, muscles are observed for lymphatic vessels under a fluorescence microscope.

The studies described in this example tested the polypeptide activity of the present invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the present invention.

(Example 52: Inhibition of TNFα-induced adhesion molecule expression by the polypeptide of the present invention)
The recruitment of lymphocytes to areas of inflammation and angiogenesis relates to specific receptor-ligand interactions between cell surface adhesion molecules (CAM) on lymphocytes and vascular endothelium. This bonding process, in both normal settings and pathological settings, cell-cell adhesion molecules in endothelial cells (EC) -1 (ICAM-1 ), vascular cell adhesion molecule -1 (VCAM-1), and inner skin leukocyte adhesion molecule-1 comprises the expression of (E- selectin), followed by a multi-stage cascade. The expression of these and other molecules in the vascular endothelium determines the efficiency with which leukocytes can adhere to the local vasculature and overflow into the local tissue during the development of an inflammatory response. Local concentrations of cytokines and growth factors are involved in regulating the expression of these CAMs.

Tumor necrosis factor α (TNF-α) (strong inflammatory-induced cytokine) is a stimulator of all three CAM in endothelial cells and participate in a wide variety of inflammatory responses obtained, often pathological Bring results.

The potential of the polypeptides of the invention to mediate suppression of TNF-α induced CAM expression can be tested. Modified ELISA assay (which uses EC as a solid phase adsorbent) using, when co stimulated with a member of a protein of the FGF family, the amount of CAM expression in TNF-alpha treatment E C Measure.

To perform this experiment, human umbilical vein endothelial cell (HUVEC) cultures were obtained from pooled cord collections and growth medium (EGM-2; Clonetics, San) supplemented with 10% FCS and 1% penicillin / streptomycin. Die
go, CA) in a humidified incubator at 37 ° C. with 5% CO ₂ . HUVECs are seeded in 96-well plates at a concentration of 1 × 10 ⁴ cells / well in EGM medium at 37 ° C. for 18-24 hours or until confluent. Subsequently, the monolayer was washed three times with a serum-free solution of RPMI-1640 supplemented with 100 U / ml penicillin and 100 mg / ml streptomycin, and for 24 hours at 37 ° C. with the specified cytokines and / or growth factors. And processed. After incubation, the cells are then evaluated for CAM expression.

Human umbilical vein endothelial cells (HUVEC) are grown to confluence in standard 96 well plates. Growth medium is removed from the cells and replaced with 90 μl of 199 medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (10 μl volume). Plates are incubated at 37 ° C. for either 5 hours (selectin and integrin expression) or 24 hours (integrin expression only). Aspirate plate, remove media and 100 μl of 0.1%
Paraformaldehyde-PBS (with Ca ++ and Mg ++) is added to each well. Hold plate at 4 ° C. for 30 minutes.

The fixative is then removed from the well and the well is PBS (+ Ca, Mg)
Wash once with + 0.5% BSA and drain. Do not let this well dry. 10 μl of diluted primary antibody is added to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin
And anti-E-selectin-Biotin at a concentration of 10 μg / ml (0.1 mg
/ Ml stock antibody at 1:10 dilution). Cells are incubated for 30 minutes at 37 ° C. in a humidified environment. Wells were washed with PBS (+ Ca, Mg
) Wash 3 times with + 0.5% BSA.

Then 20 μl of diluted ExtrAvidin-Alkaline Ph
osphotase (1: 5,000 dilution) is added to each well and 37 ° C.
Incubate for 30 minutes. Wells were PBS (+ Ca, Mg) +0.5
Wash 3 times with% BSA. 1 tablet of p-nitrophenol phosphate (p
NPP) is dissolved in 5 ml glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Triplicate standard wells were added to ExtraAvidin-Alkaline Pho in glycine buffer.
Working dilution of sphotase (1: 5,
000 (10 ⁰ )> 10 ^−0.5 > 10 ⁻¹ > 10 ^−1.5 ). 5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.
50 ng, 1.74 ng, 0.55 ng and 0.18 ng. Then 100
μl of pNPP reagent must be added to each standard well. The plate must be incubated for 4 hours at 37 ° C. 3M NaO
A volume of 50 μl of H is added to all wells. The result is quantified at 405 nm in a plate reader. The background subtraction option is used in blank wells filled with glycine buffer only. The template, concentration of the AP conjugate in each standard well to set to indicate [5.50ng; 0.18ng 1.74ng;; 0.55ng ]. Results are shown as the amount of bound AP conjugate in each sample.

The studies described in this example tested the polypeptide activity of the present invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the present invention.

Example 53: Assay for stimulation of bone marrow CD43 + cell proliferation
This assay is based on the ability of human CD34 + to grow in the presence of hematopoietic growth factors, and this assay evaluates the ability of isolated polypeptides expressed in mammalian cells to stimulate proliferation of CD34 + cells. .

The most mature precursor has previously been shown to respond to only a single signal. More immature precursors require at least two signals to respond. Thus, to test the effect of polypeptides on a wide range of progenitor cell hematopoietic activity, this assay can be performed in the presence or absence of other hematopoietic growth factors.
Contains a predetermined polypeptide. The isolated cells are combined with the test sample,
Incubate for 5 days in the presence of stem cell factor (SCF). Only SCF is bone marrow (BM
) Has a very limited effect on cell growth and acts under these conditions only as a “survival” factor. However, when combined with any factor that exhibits a stimulatory effect on these cells (eg, IL-3), SCF produces a synergistic effect. Thus, such activity can be easily detected if the tested polypeptide has a stimulatory effect on hematopoietic ancestry. Normal BM cells do not appear to detect any inhibitory effect of the desired polypeptide, or an agonist or antagonist thereof, since it has a low level of cycling cell. Therefore, assays for inhibitory effects on ancestors are preferably S
Tested in cells that are first subjected to in vitro stimulation with CF + IL + 3 and then contacted with a compound to be evaluated for inhibition of such induced proliferation.

Briefly, CD34 + cells are isolated using methods known in the art.
These cells are thawed and medium (QB with 1% L-glutamine).
SF 60 Serum-free medium (500 ml) (Quality Biology)
cal , Inc. , Gaithersburg, MD Cat # 160-20
4-101)). After some gentle centrifugation at 200 xg, the cells are allowed to stand for 1 hour. The cell number is adjusted to 2.5 × 10 ⁵ cells / ml.
During this time, 100 μl of sterile water is added to the peripheral wells of a 96 well plate. In this assay, cytokines that can be tested with a given polypeptide are 50 ng / ml rhSCF (R & D Systems, Minneapol).
is, MN, Cat # 255-SC) and 30 ng / ml r
hSCF and rhIL-3 (R & D Systems, Minneapolis,
MN, Cat # 203-ML). After 1 hour, 10 μl of prepared cytokine, 50 μl of supernatant (supernatant at 1: 2 dilution = 50 μl) and 20 μl of diluted cells are added to the medium, which is added to 100 μl final total volume. To already exist in the well. The plate is then
Place in a ° C / 5% CO ₂ incubator for 5 days.

18 hours prior to the assay, 0.5 μCi / well [
3H] thymidine is added to each well in a volume of 10 μl and the growth rate is determined. This experiment was performed using Tomtec Harvester 96 with cells each 9
Terminate by collecting from 6-well plate to filter mat. After collection, the filter mat is dried, trimmed and one OmniFilte
OmniFilt consisting of r-plate and one OmniFilter tray
er assembly. Add 60 μl Microscint to each well,
The plate is then sealed with TopSeal-A press-on sealing film. A bar code 15 sticker is secured to the first plate for counting. The sealed plate is then filled and the level of radioactivity is determined by the Packard Top C
Determine through print and collect the printed data for analysis. The radioactivity level reflects the amount of cell proliferation.

The study described in this example tests the activity of a given polypeptide that stimulates bone marrow CD34 + cell proliferation. One skilled in the art can readily modify the illustrated examples to test the activity of polynucleotides (eg, gene therapy), antibodies, agonists, and / or antagonists, and fragments and variants thereof. As a non-limiting example, antagonists that may be tested in this assay include inhibiting cell proliferation in the presence of cytokines and / or inhibiting cell proliferation in the presence of cytokines and a given polypeptide. Is expected to increase. In contrast, agonists that may be tested in this assay are expected to enhance cell proliferation and / or reduce inhibition of cell proliferation in the presence of cytokines and a given polypeptide.

The ability of a gene to stimulate the proliferation of bone marrow CD34 + cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of disorders affecting the immune system and hematopoiesis. Exemplary uses are described in the “Immune Activity” and “Infectious Diseases” sections above, and elsewhere in this specification.

Example 54: Assay for Extracellular Matrix Enhanced Cell Response (EMCR)
The purpose of the extracellular matrix enhanced cellular response (EMCRR) assay is to identify gene products (eg, isolated polypeptides) that act on hematopoietic stem cells in relation to extracellular matrix (ECM) -induced signals. .

In response to signals received from the surrounding microenvironment, cells respond to regulatory factors. For example, fibroblasts, and endothelial and epithelial stem cells are unable to replicate in the absence of signal from the ECM. Hematopoietic stem cells can undergo self-renewal in the bone marrow, but not in in vitro suspension culture. The ability of stem cells to undergo self-renewal in vitro depends on stromal cells and their interaction with the ECM protein fibronectin (fn). Cell f
Adsorption to n is α ₅ . β ₁ and α ₄ . Mediated by β ₁ integrin receptors, these receptors are expressed by human and mouse hematopoietic stem cells.
The factors that integrate in the ECM environment and are responsible for stimulating stem cell self-renewal have not yet been identified. The discovery of such factors is an important objective in gene therapy and bone marrow transplantation applications.

Briefly, a 96-well plate without polystyrene tissue culture treatment is
Coated with fn fragments at a coating concentration of 0.2 μg / cm ² . The mouse bone marrow cells are plated in serum-free culture area of 0.2ml (1,000 cells /
Well) . Cells cultured in the presence of IL-3 (5 ng / ml) + SCF (50 ng / ml) act as a positive control under conditions where stem cell self-renewal is not expected and distinct differentiation is expected. The gene product is SCF (5.0
ng / ml) with or without an appropriate negative control, where the supernatant of the test agent represents 10% of the total assay volume. The plated cells were then placed in a hypoxic environment (5% CO ₂ , 7% O ₂ and 88% N ₂
) For 7 days in a tissue culture incubator. The number of proliferating cells in the well is then quantified by measuring thymidine incorporation into the cellular DNA. Confirmation of positive hits in the assay requires characterization of the cell phenotype, which scales up the culture system,
The Ru Cormorant is achieved by using a suitable antibody reagents against cell surface antigens and FACScan.

One skilled in the art can readily modify exemplary studies for testing the activity of polynucleotides (eg, gene therapy), antibodies, agonists, and / or antagonists and fragments and variants thereof.

Particular gene product, when found to be a hematopoietic progenitor stimulant, polynucleotides and polypeptides corresponding to this gene, diagnosis of disorders that affect immune system and hematopoiesis, and may be useful in the treatment . Typical applications are the above "
It is described in the sections "Immune Activity" and "Infectious Diseases", and elsewhere herein. Gene products are also derived from stem and progenitor cells of various blood lineages (lineages).
d progenitor) and in the differentiation and / or proliferation of various cell types.

Furthermore, the polynucleotides and / or polypeptides of the gene of interest, and / or agonists and / or antagonists thereof, are also used to inhibit hematopoietic cell proliferation and differentiation, and thereby during chemotherapy It can be used to protect bone marrow stem cells from chemotherapeutic agents. This anti-proliferative effect may allow for the administration of higher doses of chemotherapeutic agents and thus more effective chemotherapeutic treatment.

In addition, polynucleotides and polypeptides corresponding to the gene of interest may also cause hematopoietic related disorders (eg, anemia, pancytopenia, leukopenia, thrombocytopenia,
Or leukemia) treatment and diagnosis. This is because stromal cells are important in the generation of hematopoietic lineage cells. These uses include bone marrow cell ex vivo culture, bone marrow transplantation, bone marrow remodeling, neoplastic radiation therapy or chemotherapy.

Example 55: Proliferation of human skin fibroblasts and aortic smooth muscle cells
The polypeptide of interest is added to a culture of normal human skin fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC), and two simultaneous assays are performed with each sample. The first assay involves normal human fibroblasts (NH
The effect of the polypeptide of interest on the proliferation of DF) or aortic smooth muscle cells (AoSMC) is tested. Abnormal proliferation of fibroblasts or smooth muscle cells is part of several pathological processes, including fibrosis and restenosis. The second assay tests IL6 production by both NHDF and SMC. IL6 production is an indicator of functional activation, activated cells have increased production of numerous cytokines and other factors, which is proinflammatory
) Or immunomodulatory results. Assay to check for costimulatory activity or inhibitory activity, simultaneously TNFa stimulation together with, and co-TNFa
Do without stimulation.

Briefly, on day 1, 96-well black plates were plated with 1000 cells / well (NHDF) or 2000 cells / well (AoS) in 100 μl culture medium.
MC). NHDF culture medium includes the following: Clonetics FB basal medium, 1 mg / ml hFGF, 5 mg / ml insulin, 50 mg / ml gentamicin, 2% FBS, while AoSMC culture medium is Cloneti
cs SM basal medium, 0.5 μg / ml hEGF, 5 mg / ml insulin,
1 μg / ml hFGF, 50 mg / ml gentamicin, 50 μg / ml A
Contains mphotericin B, 5% FBS. At least 4-5 at 37 ° C.
After incubation for a period of time, the culture medium is aspirated and replaced with growth stop medium. N
Growth stop medium for HFD contains fibroblast basal medium, 50 mg / ml gentamicin, 2% FBS, while growth stop medium for ApSMC is SM basal medium,
50 mg / ml gentamicin, 50 μg / ml Amphotericin
B, including 0.4% FBS. Incubate at 37 ° C for up to 2 days.

On the second day, serial dilutions and templates of the polypeptide of interest are designed so that they always include a media control and a known protein control. For both stimulation and inhibition experiments, the protein is diluted in growth arrest medium . For inhibition experiments, TNFa is added to a final concentration of 2 ng / ml (NHDF) or 5 ng / ml (AoSMC). Add 1/3 volume of medium containing control or supernatant and incubate until 37 days at 37 ° C./5% CO ₂ .

Transfer 60 μl from each well to another labeled 96 well plate, cover with plate sealer and store at 4 ° C. (for IL6 ELISA) until day 6.
Aseptically add Alamar Blue in an amount equal to 10% of the culture volume (10 μl) to the remaining 100 μl in the cell culture plate. 3-4 plates
Return to the incubator for hours. Then excitation at 530 nm and 590 n
Fluorescence with emission at m is measured using CytoFluor. This gives growth stimulation / inhibition data.

On Day 5, IL-6 ELISA was diluted in PBS (pH 7.4) 50-1
Perform by coating 96 well plate with 00 μl / well anti-human IL-6 monoclonal antibody and incubate overnight at room temperature.

On day 6, empty the contents of the plate into the sink and blot on a paper towel.
Prepare an assay buffer containing PBS containing 4% BSA. Plate the PB
Block with 200 μl / well Pierce Super Block blocking buffer in S for 1-2 hours, then wash buffer (PBS, 0.05%
Wash plate with Tween-20). Blot the plate on a paper towel. Subsequently, 50 μl / well of anti-human IL-6 monoclonal biotin-labeled antibody diluted to 0.50 mg / ml is added. Make dilutions in medium of IL-6 stock (30 ng / ml, 10 ng / ml, 3 ng / ml, 1 ng / ml
0.3 ng / ml, 0 ng / ml). Duplicate samples are added to the top row of the plate. Cover the plate and incubate for 2 hours at room temperature on a shaker.

Wash plate with wash buffer and blot on paper towel. EU-labeled streptavidin is diluted 1: 1000 in assay buffer and 100 μl /
Add wells. Cover the plate and incubate at room temperature for 1 hour.
Wash plate with wash buffer. Absorb on a paper towel.

Add 100 μl / well enhancement solution and shake for 5 minutes. Walla
c Read plate with DELFIA fluorometer. The readings from triplicate samples in each assay are tabulated and averaged.

A positive result in this assay suggests AoSMC cell proliferation and suggests that the gene product of interest may be involved in dermal fibroblast proliferation and / or smooth muscle cell proliferation. A positive result can also be the object of the genes / polypeptides, polynucleotides, agonist polynucleotide / polypeptide and /
Or suggest many potential uses of antagonists. For example, inflammation and immune responses, wound healing, and angiogenesis, as described throughout this specification. In particular, the polypeptide of the gene product and the polynucleotide of the gene can be used to promote wound healing and skin regeneration, and angiogenesis (both blood and lymph vessels). Vascular growth can be used, for example, in the treatment of cardiovascular disease. Further, the polypeptide of the gene product and the antagonist of the polynucleotide of the gene can act as an anti-angiogenesis (eg, anti-angiogenesis), thereby including angiogenesis, diseases, disorders, and / or It may be useful in treating the condition. These diseases, disorders, and / or conditions,
For example: malignant tumors, solid tumors, benign tumors (eg, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas); arthrosclerotic plaques
angioangiogenic diseases (eg, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal transplant rejection, neovascular glaucoma, post-lens fibroplasia, skin flushing, retinoblastoma, grapes) Meningitis and pterygium of the eye (abnormal blood vessel growth)); rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulation; hyperplastic scar (keloid); Non-union fracture
scleroderma; trachoma; vascular adhesion; myocardial angiogenesis; coronary collateral branch; cerebral collateral branch; arteriovenous malformation; ischemic limb neovascularization; Osler-We
bber syndrome; plaque neovascularization; telangiectasia; hemophilia joint; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis in the art Known and / or described herein. In addition, polypeptides of the gene product and antagonists of polynucleotides of the gene are known in the art and / or treat anti-hyperproliferative and / or anti-inflammatory diseases as described herein. Can be useful in doing so.

One skilled in the art can readily modify the exemplified studies to test the activity of polynucleotides (eg, gene therapy), antibodies, agonists, and / or antagonists, and fragments and variants thereof.

(Example 56: Expression of cell adhesion molecule (CAM) on endothelial cells)
The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAM) on lymphocytes and the vascular endothelium. The adhesion process follows a multi-stage cascade in both normal and pathological environments, which cascades intracellular adhesion molecule 1 (ICAM-1), vascular cell adhesion on endothelial cells (EC) Includes the expression of molecule 1 (VCAM-1) and endothelial leukocyte adhesion molecule 1 (E-selectin). Expression of these molecules, etc. on the vascular endothelium determines the efficiency with which leukocytes can adhere to the local vasculature and overflow into the local tissue during the progression of the inflammatory response. Local concentrations of cytokines and growth factors are involved in regulating the expression of these CAMs.

Briefly, endothelial cells (eg, human umbilical vein endothelial cells (HUVEC)) grow to confluence in standard 96 well plates. Growth medium is removed from the cells and 100 μl of 199 Medium (10% fetal bovine serum (FBS
)). Samples for testing and positive or negative controls are added to triplicate plates (10 μl volume). The plates are then incubated at 37 ° C. for either 5 hours (selectin and integrin expression) or 24 hours (integrin expression only). Plates are aspirated to remove media and 100 μl 0.1% paraformaldehyde-PBS (containing Ca ++ and Mg ++) is added to each well. Hold plate at 4 ° C. for 30 minutes. Remove fixative from wells and remove wells into PBS
Wash once with (+ Ca, Mg) + 0.5% BSA and drain. 10 μl
Of diluted primary antibody is added to the test and control wells. Anti-I
CAM-1-biotin, anti-VCAM-1-biotin, and anti-E-selectin-
Biotin is used at a concentration of 10 μg / ml (1:10 dilution of 0.1 mg / ml stock antibody). Cells are incubated at 37 ° C. for 30 minutes in a humidified environment. The wells are washed 3 times with PBS (+ Ca, Mg) + 0.5% BSA. 20 μl of diluted ExtrAvidin-Alkaline Phosp
hotase (1: 5,000 dilution, referred to herein as a practical diluent)
Is added to each well and incubated at 37 ° C. for 30 minutes. Well,
Wash 3 times with PBS (+ Ca, Mg) + 0.5% BSA. 1 tablet of p-nitrophenol phosphate (pNP) per 5 ml of glycine buffer (pH 10.4)
Dissolve P). 100 μl pNPP substrate in glycine buffer is added to each test well. A three-part standard well was added to ExtAvidi in glycine buffer.
n-Alkaline Phosphoase; 1: 5,000 (10 ⁰ )>
Prepare from a practical dilution of 10 ^−0.5 > 10 ⁻¹ > 10 ^−1.5 . 5 μl of each dilution
Is added to triplicate wells, the resulting AP content in each well is 5.5.
ng, 1.74 ng, 0.55 ng, 0.18 ng. Then 100μ
l pNNP reagent is added to each of the standard wells. The plate was heated at 37 ° C for 4
Incubate for hours. A volume of 50 μl of 3M NaOH is added to all wells. The plate is read on a 405 nm plate reader using the background removal option in blank wells filled with glycine buffer only. Furthermore, the concentration of the template, AP copolymer in each standard wells [5.5
0 ng; 1.74 ng; 0.55 ng; 0.18 ng] . Results are shown as the amount of bound AP copolymer in each sample.

Example 57: Alamar Blue Endothelial Cell Proliferation Assay
This assay can be used to quantitatively determine protein-mediated inhibition of bFGF-induced proliferation of bovine lymphatic endothelial cells (LEC), bovine aortic endothelial cells (BAEC), or human microvascular myometrium (UTMEC). This assay incorporates a fluorometric growth indicator based on detection of metabolic activity. Standard Alam
10 ng of ar Blue proliferation assay added as a source of endothelial cell stimulation
/ It is made tone in EGM-2MV, including ml of bFGF. This assay can be used with growth media and various endothelial cells with slight changes in cell concentration. Dilute the dilutions of the protein batch to be tested appropriately. medium containing no serum without bFGF to (G IBCO SFM), was used as a non-stimulated control and Angiostatin or TSP-1 are included as a known inhibitory controls.

Briefly, LEC, BAEC or UTEC can be transferred to a 96 well plate with 5
Seed growth medium at a density of 000-2000 cells / well and leave at 37 ° C. overnight. After overnight incubation of the cells, the growth medium is removed and replaced with GIBCO EC-SFM. The cells are treated in 3 wells containing additional bFGF with an appropriate dilution of the protein of interest or control protein sample (prepared in SFM) to a concentration of 10 ng / ml. Once the cells are treated with the sample, the plate (s) are returned to the 37 ° C. incubator for 3 days. 3 days later, 10 ml of stock Alamar Bl
ue (Biosource Cat # DAL1100) is added to each well and the plate (s) is returned to the 37 ° C. incubator for 4 hours. The plate or plates are then read using a CytoFluor fluorescence reader with 530 nm excitation and 590 nm emission. Direct output is recorded in relative fluorescence units.

Alamar Blue is an oxidation-reduction indicator that fluoresces and changes color in response to chemical reduction of the growth medium resulting from cell growth. As cells grow in the medium, the natural metabolic activity immediately causes a chemical reduction of the surrounding environment.
By reduction on growth, the indicator can change from an oxidized (non-fluorescent blue) form to a reduced (fluorescent red) form. That is, stimulated growth produces a stronger signal, and inhibited growth produces a weaker signal, and the total signal is proportional to the total number of cells as well as their metabolic activity. A background level of activity is observed using only starvation medium. This is compared to the output observed from the positive control sample (bFGF in growth medium) and protein dilutions.

(Example 58: Detection of inhibition of mixed lymphocyte reaction)
This assay can be used to detect and assess inhibition of mixed lymphocyte reaction (MLR) by gene products (eg, isolated polypeptides). Inhibition of MLR may result from direct effects on cell proliferation and viability, modulation of costimulatory molecules in interacting cells, modulation of adhesion between lymphocytes and accessory cells, or modulation of cytokine production by accessory cells . Multiple cells can be targeted by these polypeptides. Because the peripheral blood mononuclear fraction used in this assay is
This is because it contains T, B and natural killer lymphocytes, as well as monocytes and dendritic cells.

Polypeptides of interest found to inhibit MLR may find use in diseases related to lymphocyte and monocyte activation or proliferation. This includes asthma, arthritis, diabetes, inflammatory skin conditions, psoriasis, eczema, systemic lupus erythematosus,
Like multiple sclerosis, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, arteriosclerosis, cirrhosis, graft-versus-host disease, graft-versus-host disease, hepatitis, leukemia and lymphoma Examples include but are not limited to diseases.

Briefly, PBMC derived from a human donor is converted to Lymphocyte Separ.
ation Medium (LSM®, density 1.0 7 70 g / ml
, Organon Teknika Corporation, West Ch
Ester, PA). Purify by density gradient centrifugation using. RPMI supplemented with PBMC from two donors with 10% FCS and 2 mM glutamine
I-1640 (Life Technologies, Grand Islan
d, NY) to 2 × 10 ⁶ cells / ml. PBMC from a third donor
Is adjusted to 2 × 10 ⁵ cells / ml. 50 μl of PBMC from each donor
Add to wells of 96 well round bottom microtiter plate. Test material dilutions (50 μl) are added in triplicate to microtiter wells. The (The purpose of the protein) Test Sample 1: was added to a final dilution of 4; rhuIL-2 (R
& D Systems, Minneapolis, MN, Catalog No. 202
-IL) is added to a final concentration of 1 μg / ml; anti-CD4 mAb (
R & D Systems, clone 34930.11, catalog number MAB
379) is added to a final concentration of 10 μg / ml. Cells are 5% CO
Among _2, and cultured for 7-8 days at 37 ° C., and ^[3 H] thymidine 1 [mu] C, it is added to the wells at the end of 16 h of culture. Cells are collected and thymidine uptake
Determine using Packard TopCount. Data are expressed as the mean and standard deviation of three measurements.

Samples of the protein of interest are screened in separate experiments and negative control treatment, anti-DC4 mAB (which inhibits lymphocyte proliferation), and positive control treatment, IL-2 (recombinant or supernatant) (As this)
To promote lymphocyte proliferation).

One skilled in the art can readily modify exemplary studies to test the activity of polynucleotides (eg, gene therapy), antibodies, agonists and / or antagonists, and fragments and variants thereof.

It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

Full disclosure of each document cited in the background, detailed description and examples (including patents, patent applications, academic literature, abstracts, experimental manuals, books or other disclosures)
Are incorporated herein by reference. Furthermore, both the hard copy of the sequence listing and the corresponding computer readout form submitted with this specification are hereby incorporated by reference in their entirety. In addition, US Provisional Application No. 60 /
The contents of 164,835 and 60 / 221,142 are hereby incorporated by reference in their entirety.

ＰＴＡ−８６７
（カナダ）
出願人は、出願に基づきカナダ国特許が発行されるか、あるいは同出願が拒絶
または放棄されて回復され得なくなるかもしくは取り下げられるまでは、特許庁
長官（Ｃｏｍｍｉｓｓｉｏｎｅｒ ｏｆ Ｐａｔｅｎｔｓ）が、長官により指名
された独立の専門家に対してのみ出願中で言及された寄託済みの生物学的材料の
サンプルの供与を許可する旨を請求し、出願人は、国際出願の公表のための規則
上の準備が完了する前に、書面によりその旨を国際事務局に告知しなければなら
ない。

PTA-867
(Canada)
The applicant will be appointed by the Commissioner of the Commissioner of Patents until a Canadian patent is issued on the basis of the application or until the application is rejected or abandoned and cannot be recovered or withdrawn. Requesting that only independent specialists be allowed to provide samples of the deposited biological material referred to in the application, the applicant shall be prepared for regulatory preparation for publication of the international application. Before completion, you must notify the International Bureau in writing.

(Norway)
The applicant here only gives samples to experts in the technology until the application is published (by the Norwegian Patent Office) or is decided by the Norwegian Patent Office without publication. Request that it be. The request to this effect shall be made by the applicant to the Norwegian Patent Office before the time at which the application is made publicly available under Articles 22 and 33 (3) of the Norwegian Patent Act. If such a request is made by the applicant, the expert used in any request for the provision of a sample by a third party shall be indicated. The expert is a list of certified experts created by the Norwegian Patent Office (list of
any person listed in recognized experts), or
It can be any person approved by the applicant in individual cases.

(Australia)
Applicant here, the provision of a sample of microorganisms, prior to the grant of the patent,
Alternatively, a candidate who is a person skilled in the art who has no stake in the invention prior to lapse, rejection or withdrawal of the application.
ssee) only (notice of Australian Patent Law 3.25 (3)).

(Finland)
Applicant here is that the application is (patent and control committee (National
(Board of Patents and Regulations) that samples will only be provided to experts in the technology until they are published or decided by the National Patent and Legislative Committee without publication. ,Claim.

(UK)
The applicant here requests that the sample of the microorganism be made available only to the expert. A request to this effect must be made by the applicant to the International Bureau before the regulatory preparations for the international publication of the application are completed.

(Denmark)
The applicant here only gives samples to the expert of the technology until the application is published (by the Danish Patent Office) or is decided by the Danish Patent Office without publication. Request that it be. The request to this effect shall be made by the applicant to the Danish Patent Office before the time when the application is made publicly available under Danish Patent Act Articles 22 and 33 (3). If such a request is made by the applicant, the expert used in any request for the provision of a sample by a third party shall be indicated. The expert is a list of certified experts created by the Danish Patent Office (list of
any person listed in recognized experts), or
It can be any person approved by the applicant in individual cases.

(Sweden)
The applicant here only gives samples to the expert of the technology until the application is published (by the Swedish Patent Office) or decided by the Swedish Patent Office without publication. Request that it be. The request to this effect shall be made by the applicant to the International Bureau prior to the expiration of 16 months from the priority date (preferably PCT Applicant's Gui
Format PCT / RO / 13 described in Annex Z of volume I in de
4). If such a request is made by the applicant, the expert used in any request for the provision of a sample by a third party shall be indicated. The expert is a list of certified specialists (lis) prepared by the Swedish Patent Office.
t of recognized experts),
Alternatively, it may be any person approved by the applicant in individual cases.

(Netherlands)
Applicant here shall, until the date of issue of the Dutch patent, or until the date on which the application is rejected, withdrawn or lapsed, be subject to the provision of samples to specialists under the provisions of Patent Act 31F (1). Request that it be done only in form. The request to this effect shall be filed by the applicant before the date on which the application is made publicly available under section 22C or 25 of the Netherlands Patent Act, whichever is earlier, by the applicant. Shall be submitted to the Bureau.

Claims (1)

Invention described in the specification.