JP2002539786A - 48 human secreted proteins - Google Patents

Abstract

  (57) [Summary] The present invention relates to 48 novel human secreted proteins and isolated nucleic acids comprising the coding region of the gene encoding such proteins. Also provided are vectors, host cells, antibodies for producing human secreted proteins, and recombinant methods for making such human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating these novel human secretory protein disorders.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to newly identified polynucleotides and the polypeptides encoded by these polynucleotides, the use of such polynucleotides and polypeptides, and their production.

BACKGROUND OF THE INVENTION [0002] 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 membranes. The compartments, or organelles, bounded by each membrane contain different proteins essential for the function of that organelle. Cells use "selection signals", amino acid motifs located inside proteins, to target the protein to specific cell organelles.

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). The ER separates membrane-bound proteins from all other types of proteins. Once localized in the ER, both groups of proteins can be further directed to another organelle called the Golgi apparatus. Where the Golgi is
The protein is distributed to vesicles, including secretory vesicles, cell membranes, lysosomes, and other organelles.

[0004] 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 cell membranes 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 triggered. Similarly,
Proteins present on cell membranes are also known as "linkers" that retain proteins on the membrane.
Can be secreted into the extracellular space by proteolytic cleavage.

[0005] Despite significant progress in recent years, only a small number of 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 novel human secreted proteins and the genes encoding them. This finding allows the use of secreted proteins or the genes encoding them to detect, treat, and prevent medical disorders.

SUMMARY OF THE INVENTION [0006] The present invention relates to novel polynucleotides and their encoded polypeptides. Furthermore, the present invention relates to vectors, host cells, antibodies, and recombinant and synthetic methods for producing the polypeptides and polynucleotides.
Diagnostic methods for detecting disorders and conditions associated with the polypeptides and polynucleotides, and therapeutic methods for treating such disorders and conditions are also provided. The invention further relates to a screening method for identifying a binding partner of the polypeptide.

DETAILED DESCRIPTION Definitions The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

In the present invention, “isolated” refers to a substance that has been removed from its original environment (eg, the natural environment if it occurs naturally), It has been modified "by human hand" from its natural state. For example, an isolated polynucleotide can be part of a vector or composition of matter, or can be contained in a cell and still be "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 whole cell or mRNA preparation, genomic DNA preparation (
(Including those separated by electrophoresis and transferred to blots), sheared whole cell genomic DNA preparations, or other compositions for which the art does not exhibit the distinguishing characteristics of the polynucleotides / sequences of the present invention. I don't say anything.

In the present invention, a “secreted” protein is a protein that can be directed as a result of a signal sequence to the ER, secretory vesicle, or extracellular space, as well as being released into the extracellular space without necessarily containing the signal sequence. Refers to protein. When a secreted protein is released into the extracellular space, it can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by a number of mechanisms, including exocytosis and proteolytic cleavage.

In certain embodiments, the polynucleotide of the invention is 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 300 kb in length, 6. 200 kb, 100 kb, 50 kb, 15 kb, 10 kb,
5 kb, 5 kb, 2.5 kb, 2.0 kb or 1 kb or less. In a further embodiment, a polynucleotide of the invention, as disclosed herein, comprises a portion of a coding sequence, but does not include all or a portion of any introns. In another embodiment, a polynucleotide comprising a coding sequence does not include a coding sequence for a genomic flanking gene (ie, 5 'or 3' to the gene of interest in the genome). In other embodiments, the polynucleotides of the present invention comprise 10
00, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3,
Does not contain two or more than one genomic flanking gene coding sequences.

[0011] As used herein, "polynucleotide" refers to SEQ ID NO: X (SE
QID NO: X), or a cDNA contained in a clone deposited with the ATCC. For example, the polynucleotide may comprise a 5 'and 3' untranslated sequence, a coding region with or without a signal sequence, the nucleotide sequence of a full-length cDNA sequence including a secretory protein coding region, and fragments, epitopes, domains of the nucleic acid sequence. , And variants. Further, as used herein, “polypeptide”, when 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).
Representative clones containing all or most of the sequence for SEQ ID NO: X have been deposited with the American Type Culture Collection ("ATCC"). As shown in Table 1, each clone is identified by a cDNA clone ID (identifier) and an ATCC accession number. ATCC is a 10801 University
Boulevard, Manassas, Virginia 20110-22
09, USA. The ATCC deposit was made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure Purposes.

[0013] A "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. Contains nucleotides. “Stringent hybridization conditions” refers to 50% formamide, 5 × SSC (750 mM NaCl, 75 mM trisodium citrate),
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 Washing the filter at about 65 ° C in water.

[0014] 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 primarily achieved through manipulation of formamide concentration (lower percentages of formamide result in reduced stringency); salt conditions, or temperature. For example, lower stringency conditions may be 6 × SSPE (20 × SSPE = 3M NaCl;
_{2M NaH 2 PO 4; 0.02M EDTA} , pH7.4), 0.5% SDS
, 30% formamide, 100 μg / ml in a solution containing salmon sperm blocking DNA at 37 ° C. overnight; then 1 × SSPE, 0.1% S
Including washing at 50 ° C. with DS. Further, to achieve even lower stringency, the washes 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 replacement of alternative blocking reagents used to suppress background in hybridization experiments. Representative blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the above hybridization conditions due to compatibility issues.

Of course, a polyhybrid that hybridizes only to a polyA + sequence (eg, any 3 ′ terminal polyA + region (tract) of the cDNA shown in the sequence listing) or to a complementary stretch of T (or U) residues Nucleotides are not included in the definition of “polynucleotide”. Such a polynucleotide will hybridize to any nucleic acid molecule containing a poly (A) stretch or its complement (eg, virtually any double-stranded cDNA clone generated using oligo dT as a primer). This is because they soy.

[0017] The polynucleotides of the present invention can be composed of any polyribonucleotide or polydeoxyribonucleotide, including unmodified RNA or unmodified DN.
A or modified RNA or DNA. For example, polynucleotides are single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions.
, Single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, single-stranded, or more typically a mixture of double-stranded or single- and double-stranded regions. It can be composed of hybrid molecules, including possible DNA and RNA. In addition, the polynucleotide may be composed of triple stranded regions, including RNA or DNA or both RNA and DNA. Polynucleotides can also include one or more modified bases or DNA or RNA backbones that have been 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 can be composed of amino acids linked together by peptide bonds or modified peptide bonds, ie, peptide isosteres, and include amino acids other than the 20 gene-encoded amino acids. May be included. The polypeptide can be produced by a natural process, such as post-translational processing, or by chemical modification techniques well known in the art.
Can be modified. Such modifications can be found in basic texts, as well as in more detailed research papers,
As well as in many research literatures. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. It is understood 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 may 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. Modifications include:
Acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin,
Covalent attachment of heme moieties, nucleotides or nucleotide derivatives, lipids or lipid derivatives, phosphatidylinositol (phosphot)
covalent bonds, cross-linking, cyclization, disulfide bond formation,
Demethylation, formation of covalent crosslinks, formation of cysteine, formation of pyroglutamic acid, 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, sulphation, arginylation, and ubiquitination. (For example, PROTEI
NS-STRUCTURE AND MOLECULAR PROPERTIER
S, 2nd edition, T.S. E. FIG. Creighton, W.M. H. Freeman and
Company, New York (1993); POSTTRANSLATI
ONAL COVALENT MODIFICATION OF PROTEI
NS, B .; C. Edited by Johnson, Academic Press, New Y
ork, pp. 1-12 (1983); Seifter et al., Meth Enzymo.
l 182: 626-646 (1990); Rattan et al., Ann NYA.
cad Sci 663: 48-62 (1992)).

“SEQ ID NO: X (SEQ ID NO: X)” refers to a polynucleotide sequence, while “SEQ ID NO: Y (SEQ ID NO: Y)” refers to a polypeptide sequence, and both sequences , Identified by the integers specified in Table 1.

A “biologically active polypeptide” refers to a polypeptide of the present invention (including the mature form, as measured by a particular biological assay, with or without dose dependence). And the like, but not necessarily the same. If there is a dose dependency, it need not be the same as the dose dependency of the polypeptide, but rather is substantially similar to the dose dependency on a given activity when compared to the polypeptide of the present invention ( That is, the candidate polypeptide is
Shows greater activity or about 1/25 as compared to the polypeptides of the invention.
Or more, and preferably about 1/10 or more, and most preferably about 1/3.
The above activities are shown).

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

Such regions can cause a database similarity search program such as BLAST to find a high-scoring sequence match that does not imply true homology. Most weight matrices (used to score alignments generated by BLAST) are generally of the group of hydrophobic amino acids (L, V and I) found in certain low complexity regions. The problem is exacerbated by the fact that giving a match between any of them gives a high score almost as high as an exact match.

In order to correct this, BLASTX. 2 (version 2.0a5MP-
WashU) "masks" lower complexity in certain sequences.
) "Using two filters (" seg "and" xnu "). These filters analyze the sequence for such regions and create new sequences in which amino acids in lower complexity regions have been replaced by the letter "X". This is then the input sequence to the BLASTX program (sometimes referred to herein as "
Query and / or "Q"). While this regimen helps to ensure that high-scoring matches represent true homology, the negative result is that the BLASTX program uses the query sequence masked by the filter to draw the alignment. Exists.

Thus, the stretch of “X” in the alignment shown in the applications below 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 the sequence is, or is not, identical to the sequence disclosed in the alignment of the invention. Such a stretch may simply indicate that, as defined above, the BLASTX program masks amino acids in the region of low complexity due to detection of the region. In all cases, the reference sequences shown herein, in the sequence listing (Table 1), and / or in the deposited clones (sometimes herein referred to as “Subject”, “Sbjct”) , And / or "S") are crucial embodiments of the invention and, unless stated otherwise in detail herein, relative to the partial sequence shown in the alignment, It should not be construed as limiting the invention.

(Polynucleotides and polypeptides of the invention) (Characteristics of the protein encoded by Gene No. 1) This gene was obtained from the following tissue / cDNA library: Soares reti
Na2b4HR is predominantly expressed and to a lesser extent H. F
rotational cortex, epileptic, re-excision;
Morton Fetal Cochlea; Synovial IL-1 / T
NF stimulated; Alzheimers, spongy chan
Ge; expressed in Hodgkin's Lymphoma II.

[0026] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 11 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 832 of SEQ ID NO: 11, and b is 15 to 8
An integer of 46, wherein both a and b correspond to the nucleotide residue positions set forth in SEQ ID NO: 11, and wherein b is a + 14 or greater, excluding one or more polynucleotides You.

(Characteristics of the protein encoded by Gene No. 2) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gi | 38458 (in which “ PTB-associated splitting factor [Ho
mo sapiens] (all information available through the cited accession numbers is hereby incorporated by reference) and has determined to share sequence homology with the sequences available through. . A partial alignment demonstrating the observed homology is shown immediately below.

[0028]

Embedded image The gi | 38458 segment shown above as "Sbjct" is shown as the sequence of SEQ ID NO: 107. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein.

[0029] Preferred polypeptides of the invention are those that are queried in the alignments set forth above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 108 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Soares feta
soliver melanocyte 2NbHM; Soars melanocyte 2NbHM; Soars melanocyte 2NbHM;
es retina N2b4HR; Soares planta Nb2
HP; Soares infant brain 1NIB; Human T-
Cell Lymphoma; Human Amygdala; Human B
Cell Lymphoma; Soares_fetal_lung_NbH
L19W; HSA 172 Cells; Soares_pregnant_u
terus_NbHPU; Human Cerebellum; Soares_
fetal_heart_NbHH19W; Human Whole Brai
n # 2-Oligo dT> 1.5 Kb; Soares_multiple_
sclerosis_2NbHMSP; Human umbilical ve
in endothial cells, IL-4 induced; UL
Cerative Colitis; Bone Marrow Stromal
Cell, untreated; Human Adrenal Ground
Tumor; Stratagene HeLa cell s3 937216
H .; Stratagene hNT neuron (# 937233); Fr
total cortex, epileptic, re-excision; S
Tratagene lung (# 937210); Soares_pregn
ant_uterus_NbHPU; Soares_fetal_liver_
H. spray_1 NFLS_S1; Adult Spring, ziplo
x; Human Resting Macrophage; Human Fet
al Liver, mixed 10 & 14 week; Human Pr
ostate, subtracted; Striatum Depressio
n; Morton Fetal Cochlea; A1-Cell Line;
Human Tonsils, Fraction 2; Human Adult
Spleen; Skin; Human Pancreatic Carcin
oma; NTERA2 teratocarcinoma cell line
+ Retinoic acid (14 days); Pancreas Tum
or PCA4 Tu; Human Colon Cancer, re-exc
ision; pBMC stimulated w / poly I / C; Smo
human muscle, IL1b induced; human corpus
colosum; H Female Blader, Adult; Soar
es_total_fetus_Nb2HF8_9w; Healing gro
in Wound, 7.5 hours post incision; NTE
RA2 + retinoic acid, 14 days; Jurkat TC
cell G1 phase; Myloid Progenitor Cell
Line; Soares_parathyroid_tumor_NbHPA
; Human Ovary; Human Manic Depression
Tissue; Fetal Liver, subtraction II;
Kidney Medulla, re-excision; T-Cell PH
A 16 hrs; Human Bone Marrow, re-excisi
on; Human Brain, Striatum; Human Osteob
lasts II; Human Uterine Cancer; Human
Rhabdomyosarcoma; Synovial Fibroblast
s (control); Stratagene colon (# 937204)
Human Thmus Stromal Cells; Stratage
ne muscle 937209; Colon Normal II; bre
ast lymph node cDNA library; Soares_m
multiple_sclerosis_2NbHMSP; Bone marro
w; Human Adult Pulmonary, re-excision;
T Cell helper I; Activated T-Cell (12h
s) / Thiouridine lined Eco; Endotheli
al cells-control; Human Microvasular
Endothelial Cells, fracture. A: CD34 posi
five cells (Cord Blood); Spring, Chroni
activated lymphocytic leukemia; Activated TC
ell (12h) / Thiourdine-re-excision;
an Testes; Bone Marrow Cell Line (RS4,
11); Human Endometrial Tumor; Hodgkin '
s Lymphoma II; Keratinocyte; Human 8W
eeek Whole Embryo; Nine Week Old Early
Stage Human; Soares_sensent_fibrob
lasts_NbHSF; Primary Dendritic Cells,
lib1 was found to be expressed.

Preferred epitopes include those comprising the sequence shown in SEQ ID NO: 60 as residues Pro-17 to Ala-27.

[0032] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 12 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1420 of SEQ ID NO: 12, and b is 15 to
An integer of 1434, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 12, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of the protein encoded by Gene No. 3) Using the computer algorithm BLASTX, the translation product of this gene may be identified by the following database entry number gi | 171216 (
It is described as "unknown [Homo sapiens]" therein.)
(All information available through the cited accession numbers is incorporated herein by reference) and has determined to share sequence homology with the sequences available through.
A partial alignment demonstrating the observed homology is shown immediately below.

[0034]

Embedded image The segment of gi | 171216, shown above as “Sbjct”,
It is shown as a sequence of SEQ ID NO: 109 and / or SEQ ID NO: 111.

[0035] Preferred polypeptides of the present invention may be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 110 and / or SEQ ID NO: 112 corresponding to the sequence.

[0036] This gene is Frontal cortex, epileptic, r
It is mainly expressed in e-excision.

[0037] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 13 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 747 of SEQ ID NO: 13, and b is 15 to 7
An integer of 61, wherein both a and b correspond to the nucleotide residue positions set forth in SEQ ID NO: 13, and wherein b is a + 14 or greater), excluding one or more polynucleotides You.

(Characteristics of Protein Encoded by Gene No. 4) This gene was obtained from the following tissue / cDNA library: Soares mela
nocite 2NbHM; Soares infant brain 1NI
B, and to a lesser extent Soares_NhHM
Pu_S1; Colon Normal II; Soares plant
a Nb2HP; Osteoblastoma-normalized B; H
human Gall Blader, fraction II; Human
Lung; Human T-cell lymphoma, re-excisi
on; H Female Blader, Adult; 12 Week Ol
d Early Stage Human, II; Rejected Kidn
ey, lib 4; Frontal cortex, epileptic
, Re-excision; 12 Week Old Early Stage
Human; Human Osteoblastoma; Soares_mu
<tb>_type_sclerosis_2NbHMSP; Human Amygd
ala; Human Testes; Human Endometrial T
umor; expressed in Human Cerebellum.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 14 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 427 of SEQ ID NO: 14, and b is 15 to 4
An integer of 41, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 14, and wherein b is a + 14 or greater, excluding one or more polynucleotides You.

(Characteristics of the protein encoded by Gene No. 5) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gi | 184435 (
The sequence homology to the sequences available through the "SH protein [Mumps virus]" therein (all information available through the cited accession number is hereby incorporated by reference). Decided to share. A partial alignment demonstrating the observed homology is shown immediately below.

[0041]

Embedded image The segment of gi | 1840435, shown above as "Sbjct",
This is shown as the sequence of SEQ ID NO: 113.

Preferred polypeptides of the present invention can be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 114 corresponding to the sequence.

This gene is derived from H. Frontal cortex, epileptic, r
It is mainly expressed in e-excision.

[0044] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 15 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 438 of SEQ ID NO: 15, and b is 15 to 4
52, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 15, and wherein b is a + 14 or greater), excluding one or more polynucleotides You.

(Characteristics of Protein Encoded by Gene No. 6) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gnl | PID | e12 as a non-limiting example:
89133 (in which "SPR2G protein [Mus musculus
s] ") (all information available through the accession number cited is hereby incorporated by reference herein) and shares sequence homology with the sequence available. A partial alignment demonstrating the observed homology is shown immediately below.

[0046]

Embedded image The segment of gnl | PID | e128133 shown above as “Sbjct” is shown as the sequence of SEQ ID NO: 115. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein.

The preferred polypeptides of the present invention can be queried in the alignment shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 116 corresponding to the sequence.

This gene was found to be expressed primarily in the Human Fetal Kidney.

Preferred epitopes include residues Gln-21 to Gly-27, Lys-3
5-Tyr-42 includes an epitope containing the sequence shown in SEQ ID NO: 64.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 16 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 2487 of SEQ ID NO: 16, and b is 15 to
An integer of 2501, where both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 16, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 7) This gene was obtained from the following tissue / cDNA library: Stomach can
cer (human), re-excision; Frontal cor
tex, epileptic, re-excision; Human Amyg
dala; Soares fetal river spring 1NFLS
It was found to be mainly expressed in.

[0052] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 17 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 1598 of SEQ ID NO: 17, and b is 15 to
1612, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 17, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of the protein encoded by Gene No. 8) Using the computer algorithm BLASTX, the translation product of this gene was identified as a non-limiting example by the following database accession number gi | 168787 (wherein “ conditionation-specific protein con
-10 [Neurospora crassa]) (all information available through the cited accession number is hereby incorporated by reference herein) and shares sequence homology with the sequence available. It was determined. A partial alignment demonstrating the observed homology is shown immediately below.

[0054]

Embedded image The segment of gi | 168787, shown above as "Sbjct", is shown as the sequence of SEQ ID NO: 117.

[0055] Preferred polypeptides of the invention are those that are queried in the alignment shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 118 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Soares_multit
ip_sclerosis_2NbHMSP is expressed predominantly and, to a lesser extent, Soares infant brain 1NIB; S
Soinals_NhHMPU_S1; Heart;
hypothalamus, frac A, re-excision; pros
state-edited; Human Adult Retina; Atr
opic Endometrium; Stratagene ovary (#
937217); Human Prostate Cancer, Stage
C fraction; H Female Blader, Adult; Hu
manic Depression Tissue; Soares_s
escent_fibroblasts_NbHSF; Soares br
east 2NbHBst; Human Whole Six Week Ol
d Embryo; Frontal cortex, epileptic,
re-excision; Human Testes Tumor; Human
Fetal Heart; NCI_CGAP_Lu5; NCI_CGAP_K
id3; WATM1; Human Adult Pulmonary, re-e
It was found to be expressed in xcision.

[0057] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 18 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 2721 of SEQ ID NO: 18, and b is 15 to
An integer of 2735, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 18, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 9) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gnl | PID | e12 as a non-limiting example:
86151 (in which "hypothetical protein [Arab
idopsis thaliana]) (all information available through the accession number cited is hereby incorporated by reference) and has determined to share sequence homology with the sequences available through it. . A partial alignment demonstrating the observed homology is shown immediately below.

[0059]

Embedded image The segment of gnl | PID | e128151 shown above as “Sbjct” is shown as the sequence of SEQ ID NO: 119.

[0060] Preferred polypeptides of the present invention can be queried in the alignments set forth above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 120 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Human Pancr
H. Eatic Carcinoma; Frontal cortex, epi
leptic, re-excision; Soares placenta N
b2HP; Soares fetal river spring 1NFLS
It was found to be mainly expressed in.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 19 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 949 of SEQ ID NO: 19, and b is 15 to 9
An integer of 63, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 19, and wherein b is a + 14 or greater, excluding one or more polynucleotides You.

(Characteristics of Protein Encoded by Gene No. 10) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number pir | A03277 |
SMHO1A (in which "metallothionein 1A-horse"
) (All information available through the accession number cited is hereby incorporated by reference herein) and has determined to share sequence homology with the sequences available. A partial alignment demonstrating the observed homology is shown immediately below.

[0064]

Embedded image The segment of pir | A03277 | SMHO1A shown above as “Sbjct” is shown as the sequence of SEQ ID NO: 121. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein.

[0065] Preferred polypeptides of the present invention can be queried in the alignment shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence set forth as SEQ ID NO: 122 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Spinal cord
H .; Frontal cortex, epileptic, re-excis
was found to be expressed predominantly in Ion.

[0067] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 20 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1647 of SEQ ID NO: 20, and b is 15 to
1661, where both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 20, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 11) Frontal cortex, epileptic, r
It was found to be predominantly expressed in e-excision.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 21 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 519 of SEQ ID NO: 21, and b is 15 to 5
33, wherein both a and b correspond to the nucleotide residue positions set forth in SEQ ID NO: 21, and wherein b is a + 14 or greater, excluding one or more polynucleotides You.

(Characteristics of the Protein Encoded by Gene No. 12) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gi | 4151809 (as a non-limiting example)
Among them, "(AF102855) synaptic SAPAP-intera"
cting protein Synamon [Rattus norvegi
cus]] (all information available through the accession number cited is hereby incorporated by reference herein) and shares sequence homology with the sequence available. A partial alignment demonstrating the observed homology is shown immediately below.

[0071]

Embedded image The segment of gi | 4151809, shown above as "Sbjct",
This is shown as the sequence of SEQ ID NO: 123. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art, some of which are described elsewhere herein. Assays to determine such activity are also
It is known in the art, some of which are described elsewhere herein.

[0072] Preferred polypeptides of the present invention can be queried in the alignment shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 124 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Soares feta
It is expressed predominantly in lliversplen 1NFLS and, to a lesser extent, H. Frontal cortex, epileptic, r
e-excision; Macrophage-oxLDL, re-excis
ion; Colon Carcinoma; Soares ovary tum
or NbHOT; Human Kidney Cortex, re-resc
ue; Pancreas Tumor PCA4 Tu; Human Whol
e Brain # 2-Oligo dT> 1.5 Kb; NCI_CGAP_G
CB1; Stratagene NT2 neuronal precurso
r 937230; Human Amygdala; Soares_fetal
_Heart_NbHH19W; Monocyte activated; So
ares infant brain 1NIB; Brain, normal;
Human Fetal Heart, Differential (Fetal
-Specific); Human Whole 6 Week Old Em
bryo (II), subt; Apoptotic T-cell, re-ex
cision; Breast Cancer cell line, MDA 3
6; Fetal Liver, subtraction II; Stratag
ene fetal splen (# 937205); Human Feta
l Dura Mater; NCI_CGAP_Br2; NCI_CGAP_L
u5; Human Adult Tests, Large Inserts,
Reexcision; Human Activated Monocytes
Macrophage (GM-CSF treated); Stratage
ne colon (# 937204); NCI_CGAP_Br3; NCI_C
GAP_GCB1; Human Testes Tumor; H Macrop
hage (GM-CSF treated), re-excision; Col
on Tumor II; human tonsils; Endothelia
l cells-control; Human Microvascular
Endothelial Cells, fracture. A; neutrophil
s control; Nine Week Old Early Stage
Human; was found to be expressed in Human Cerebellum.

Preferred epitopes include those comprising the sequence shown in SEQ ID NO: 70 as residues Ser-34 to Arg-45.

[0075] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 22 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1847 of SEQ ID NO: 22, and b is 15 to
1861, where both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 22, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 13) Using the computer algorithm BLASTX, the translation product of this gene may be identified by the following database entry number pir | A21124 |
A21124 (in which "Sxr (Bkm-homolog) sex-dete
(described as “rming region protein-mouse”) (all information available through the cited accession number is incorporated herein by reference) to share sequence homology with the sequence available. Were determined. A partial alignment demonstrating the observed homology is shown immediately below.

[0077]

Embedded image The segment of pir | A21124 | A21124 shown above as "Sbjct" is shown as the sequence of SEQ ID NO: 125. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein.

[0078] Preferred polypeptides of the present invention can be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 126 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: CD34 + cell,
I; HSA 172 Cells; Frontal cortex, epi
Leptic, re-excision; Anagic T-cell; CD
It was found to be predominantly expressed in 34 positive cells (Cord Blood).

Tissue distribution in immune cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosing and treating various immune system disorders. Representative uses are described in Examples 11, 13, 14, 16, 18, 19, 20, and 27 and "immunological activity" and "sensitivity disorders" elsewhere herein.
Described in Briefly, this expression indicates a role in regulating the growth, survival, differentiation, and / or activation of hematopoietic cell lines, including blood stem cells. Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests utility for treating cancer (eg, by boosting the immune response).
Expression in cells of lymphoid origin indicates that this natural gene product is involved in immune function. Thus, it is also arthritic, asthmatic, immunodeficient diseases such as AIDS, leukemia, rheumatoid arthritis, chronic granulomatosis, inflammatory bowel disease, sepsis, contusion, neutropenia, neutropenia, psoriasis Hypersensitivity (eg, T cell mediated cytotoxicity); immune response 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, 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 properties of other blood cells or recruits hematopoietic cells to the site of injury. Accordingly, this gene product is believed to be useful in the expansion of stem cells and related progenitor cells of various blood lineages, and in the differentiation and / or expansion of various cell types. In addition, the protein may also be used to determine biological activity, elicit antibodies (as tissue markers), isolate relevant ligands or receptors, identify agents that modulate their interactions, and Can be used as a nutritional supplement. The proteins, as well as antibodies against the proteins, may exhibit utility as tumor markers and / or immunotherapeutic targets for the tissues listed above.

[0081] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 23 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1-2630 of SEQ ID NO: 23, and b is 15-
An integer of 2644, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 23, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of the protein encoded by Gene No. 14) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number sp | Q29909 | Q
29909 (described therein as "Glutaminese (Fragment)") (all information available through the cited accession number is incorporated by reference herein) and the sequence homology with the sequence available through Decided to share. A partial alignment demonstrating the observed homology is shown immediately below.

[0083]

Embedded image The segment of sp | Q29009 | Q29009 indicated above as “Sbjct” is shown as the sequence of SEQ ID NO: 127 and / or 129. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein.

[0084] Preferred polypeptides of the present invention can be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 128 and / or SEQ ID NO: 130 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Soares infa
nt brain 1 NIB, and to a lesser extent Human T-Cell Lymphoma; Nine Week Old
Early Stage Human; Human Cerebellum;
Human Amygdala, re-excision; Frontal
cortex, epileptic, re-excision; Soares
melanocyte 2NbHM; Human Adult Pulmon
ary, re-excision; Monocyte activated; H
odgkin's Lymphoma II; Soares fetal li
ver spleen 1NFLS; Human Fetal Brain, n
normalized AC5002; Human Striatum Depr
ession, re-rescue; Human Infant Adrena
l Grand, Subtracted; H Umbilical Vein
Endothelial Cells, frac A, re-excision
Human colon cancer; Human adult small
l intestine, re-excision; Human Tonsil
s, Lib 2; Synovial IL-1 / TNF stimulated
Human Umbilical Vein, Endo. remake; Hu
man Osteosarcoma; Human Manic Depress
ion Tissue; Spinal Cord, re-excision; H
human Brain, Striatum; Human Hippocampu
s; Resting T-Cell Library, II; Human Su
bstantia Nigra; Dendritic cells, pool
d; Primary Dendritic cells, frac 2; hum
an tonsils; T Cell helper I; Human Ost
eoblastoma; Human Amygdala; Smooth mus
cle, control; Primary Dendritic Cells,
lib1 was found to be expressed.

[0086] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 24 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 3101 of SEQ ID NO: 24, and b is 15 to
An integer of 3115, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 24, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 15) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gnl | PID | e12 as a non-limiting example:
89131 (in which "SPR2F protein [Mus musculu
s] ") (all information available through the accession number cited is hereby incorporated by reference herein) and shares sequence homology with the sequence available. A partial alignment demonstrating the observed homology is shown immediately below.

[0088]

Embedded image The segment of gnl | PID | e1289131 shown above as “Sbjct” is shown as the sequence of SEQ ID NO: 131. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art, some of which 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 present invention can be queried in the alignment shown above (gaps introduced into the sequence by computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 132 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Soares plaque
enta Nb2HP is predominantly expressed and, to a lesser extent,

[0091]

Embedded image Was found to be expressed in

Preferred epitopes include residues Met-1 to Arg-7, Ala-70 to
Ser-77 includes an epitope including the sequence shown in SEQ ID NO: 73.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 25 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 979 of SEQ ID NO: 25, and b is 15 to 9
An integer of 93, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 25, and wherein b is a + 14 or greater, excluding one or more polynucleotides You.

(Characteristics of Protein Encoded by Gene No. 16) Using the computer algorithm BLASTX, the translation product of this gene may be identified by the following database entry number pir | A48203 |
A48203 (in which "interleukin 14 precursor"
(Described as “human”) (all information available through the cited accession numbers is incorporated herein by reference) and has determined to share sequence homology with the sequences available through. A partial alignment demonstrating the observed homology is shown immediately below.

[0095]

Embedded image The segment of pir | A48203 | A48203 shown above as “Sbjct” is shown as the sequence of SEQ ID NO: 133. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein.

[0096] Preferred polypeptides of the present invention can be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 134 corresponding to the sequence.

This gene is Frontal cortex, epileptic, r
It was found to be predominantly expressed in e-excision.

[0098] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 26 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1066 of SEQ ID NO: 26, and b is 15 to
An integer of 1080, wherein both a and b correspond to the nucleotide residue positions set forth in SEQ ID NO: 26, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 17) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database accession number gi | 171216 (
It is described as "unknown [Homo sapiens]" therein.)
(All information available through the cited accession numbers is incorporated herein by reference) and has determined to share sequence homology with the sequences available through.
A partial alignment demonstrating the observed homology is shown immediately below.

[0100]

Embedded image The segment of gi | 171216, shown above as “Sbjct”,
This is shown as the sequence of SEQ ID NO: 135.

Preferred polypeptides of the present invention can be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 136 corresponding to the sequence.

This gene is Frontal cortex, epileptic, r
It was found to be predominantly expressed in e-excision.

[0103] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 27 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1685 of SEQ ID NO: 27, and b is 15 to
An integer of 1699, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 27, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 18) This gene was obtained from the following tissue / cDNA library: Soares infa
nt brain 1 NIB is predominantly expressed and, to a lesser extent, Human Neutrophil, Activated; Soares_m
multiple_sclerosis_2NbHMSP; Human Sole
us; Kidney Cortex, subtracted; Human
Frontal Cortex, Schizophrenia; Jurkat
T. Cell, S phase; Frontal cortex, epi
leptic, re-excision; Brain frontal cor
tex; H Macrophage (GM-CSF treated), re-
excision; Neutrophils control, re-exci
sion; Human Amygdala; Human Cerebellum
Was found to be expressed in

Preferred epitopes include those comprising the sequence shown in SEQ ID NO: 76 as residues Thr-26 to Ala-31.

[0106] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 28 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1405 of SEQ ID NO: 28, and b is 15 to
An integer of 1419, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 28, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of the protein encoded by Gene No. 19) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gi | 182221 (in which “ Sharing sequence homology with sequences available through ORF 3 [Homo sapiens]) (all information available through the cited accession numbers is hereby incorporated by reference). It was determined. A partial alignment demonstrating the observed homology is shown immediately below.

[0108]

Embedded image The segment of gi | 182221 shown above as “Sbjct” is shown as the sequence of SEQ ID NO: 137.

[0109] Preferred polypeptides of the present invention can be queried in the alignment shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence set forth as SEQ ID NO: 138 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Soares reti
na N2b4HR; Soares melanocyte 2NbHM; So
are mainly expressed in ares planta Nb2HP, and to a lesser extent, Human Whole 6 Week Old Embryo
(II), subt; Hypothalamus; Human (Caco-2)
cell line, adenocarcinoma, colon, remark
e; Salary Valley; Stratagene fetal re
tina 937202; Stratagene endothelial c
ell 937223; Human Frontal Cortex, Chi.
zophrenia; Healing groin wound, 6.5 ho
urs post incision; Wilm's tumor; Human
Prostate; Human Hypothalmus, Schizoph
renia; Synovial Fibroblasts (control);
Human Tests Tumor; Soares_fetal_lung
_NbHL19W; Human Synovial Sarcoma; huma
n tonsils; Endothelial cells-control;
Anagic T-cell; Human Osteoblastoma; T
cell helper II; Primary Dendritic Ce
lls, lib1 was found to be expressed.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 29 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 941 of SEQ ID NO: 29, and b is 15 to 9
55, where both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 29, and where b is a + 14 or greater), excluding one or more polynucleotides You.

(Characteristics of Protein Encoded by Gene No. 20) This gene was found to be mainly expressed in Human Fetal Spring.

Preferred epitopes include those comprising the sequence shown in SEQ ID NO: 78 as residues Pro-36 to Arg-41.

[0114] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 30 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 864 of SEQ ID NO: 30, and b is 15 to 8
An integer of 78, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 30, and wherein b is a + 14 or greater), excluding one or more polynucleotides You.

(Characteristics of the protein encoded by Gene No. 21) Frontal cortex, epileptic, r
It was found to be predominantly expressed in e-excision.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 31 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 2085 of SEQ ID NO: 31, and b is 15 to
2099, where both a and b correspond to the nucleotide residue positions set forth in SEQ ID NO: 31, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of the Protein Encoded by Gene No. 22) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database accession number gnl | PID | e13 as a non-limiting example:
63948 (in which "(AJ010045) Guanine Nucleot
(described as "ide-exchange factor [Mus musculus]") (all information available through the cited accession number is shared by the sequence available through the reference numbers). Decided to. A partial alignment demonstrating the observed homology is shown immediately below.

[0118]

Embedded image The segment of gnl | PID | e136948 shown above as "Sbjct" is shown as a sequence of SEQ ID NO: 139. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art, some of which are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein.

[0119] Preferred polypeptides of the present invention can be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence set forth as SEQ ID NO: 140 corresponding to the sequence.

When tested against the K562 leukemia cell line, supernatants taken from cells containing this gene activated the ISRE assay. Therefore, this gene is Jak-
It appears to activate leukemia cells through the STAT signaling pathway. This interferon-sensitive response element (interferon-sensit)
The live response element is a promoter element found upstream of many genes involved in the Jak-STAT pathway. This Ja
The k-STAT pathway is a large signaling pathway involved in cell differentiation and proliferation. Thus, activation of the Jak-STAT pathway, reflected in ISRE element binding, may be used to indicate proteins involved in cell growth and differentiation.

This gene was obtained from the following tissue / cDNA library: Human T-Cell
Lymphoma; Soares fetal river spring
It is predominantly expressed in 1NFLS and, to a lesser extent, Pancrea
s Islet Cell Tumor; Human Testes; Huma
n Tonsils, Lib 2; Human Placenta; Soare
s_fetal_liver_splen_1 NFLS_S1; Monocy
te activated; Nine Week Old Early Sta
geo Human; Soares_parathyroid_tumor_Nb
HPA; Primary Dendritic Cells, lib 1; So
ares infant brain 1NIB; Morton Fetal
Cochlea; Epididymus, cauda; Human Lu
ng; Soares retina N2b4HR; Whole Brai
n # 2, re-excision; Human Synovium; Huma
n Colon Cancer, re-excision; pBMC stim
ultated w / poly I / C; Human Umbilical Ve
in, Endo. remake; Salaryy Grand, Lib 2;
Human Infant Brain; Soares_pineal_gl
and_N3HPG; Fetal Liver, subtraction II
Human Fetal Dura Mater; Human Pancre
as Tumor; T-Cell PHA 24 hrs; Human Adi
pose; Rejected Kidney, lib 4; Soares_Nh
HMPU_S1; Resting T-Cell Library, II; 12
Week Old Early Stage Human; Human Su
bstantia Nigra; Soares_multiple_scler
osis_2NbHMSP; Human Fetal Heart; human
tonsils; Activated T-Cell (12hs) / Thio
uridine labelledEco; Human Microvascu
lar Endothelial Cells, fracture. A: Human
B Cell Lymphoma; Human Bone Marrow, tr
eated; Activated T-cell (12h) / Thiourid
ine-re-excision; Human Endometrial Tu
mor; T cell helper II; Human Cerebellu
m.

The cell distribution in immune cells and the supernatant taken from cells containing this gene was
The fact that it activated the ISRE assay indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosing and treating various immune system disorders. Representative uses are described in Examples 11, 13, 14, 16, 18, 19,
20, 27, and elsewhere herein, "immunological activity"
Infectious diseases ". Briefly, this expression indicates a role in regulating the growth, survival, differentiation, and / or activation of hematopoietic cell lines, including blood stem cells. Involvement in the regulation of cytokine production, antigen presentation, or other processes has utility for the treatment of cancer (eg, by boosting the immune response)
Suggests. Expression in cells of lymphoid origin indicates that this natural gene product is involved in immune function. Thus, it is also arthritic, asthmatic, immunodeficient diseases such as AIDS, leukemia, rheumatoid arthritis, chronic granulomatosis, inflammatory bowel disease, sepsis, contusion, neutropenia, neutropenia, psoriasis Hypersensitivity (eg, T cell mediated cytotoxicity); immune response 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, 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 properties of other blood cells or recruits hematopoietic cells to the site of injury. Accordingly, this gene product is believed to be useful in the expansion of stem cells and related progenitor cells of various blood lineages, and in the differentiation and / or expansion of various cell types. In addition, the protein may also be used to determine biological activity, elicit antibodies (as tissue markers), isolate relevant ligands or receptors, identify agents that modulate their interactions, and Can be used as a nutritional supplement. The proteins, as well as antibodies against the proteins, may exhibit utility as tumor markers and / or immunotherapeutic targets for the tissues listed above.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 32 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 2732 of SEQ ID NO: 32, and b is 15 to
An integer of 2746, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 32, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 23) This gene was obtained from the following tissue / cDNA library: Human Chond
rosarcoma, and to a lesser extent,

[0125]

Embedded image Was found to be expressed in

[0126] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 33 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1427 of SEQ ID NO: 33, and b is 15 to
An integer of 1441 wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 33, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of the protein encoded by Gene No. 24) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number pir | JL0067 |
JL0067 (in which "Met-enkephalin-Arg-Phe-p
ig ") (all information available through the accession number cited is hereby incorporated by reference herein) and shares sequence homology with the sequence available. A partial alignment demonstrating the observed homology is shown immediately below.

[0128]

Embedded image The segment of pir | JL0067 | JL0067 shown above as "Sbjct" is shown as the sequence of SEQ ID NO: 141. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art, some of which 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 present invention include those having the amino acid sequence shown as SEQ ID NO: 142, corresponding to the Query sequence, in the alignment shown above (gaps introduced into the sequence by the computer are of course removed). .

This gene was obtained from the following tissue / cDNA library: Human Lung
Cancer, re-excision; Stomach cancer (hu
man), predominantly expressed in re-excision, and to a lesser extent Human Hypothalamus, schizophrenia.
, Re-excision; Synovial hypoxia; Spinal
Cord, re-excision; Temporal cortex-Al
zheizmer, subtracted; Macrophage-oxLDL
, Re-excision; T cell helper II; Larynx
Carcinoma; Whole 6 Week Old Embryo; S
ynovial hypoxia-RSF subtracted;
s tumor; Brain Frontal Cortex, re-exci
session; 12 Week Old Early Stage Human, I
I; Human Fetal Dura Mater; Synovial Fi
broblasts (control); Frontal cortex,
epileptic, re-excision; Human T-Cell L
ymphoma; Primary Dendritic cells, frac
2; Soares_fetal_heart_NbHH19W; Soares
_Multiple_sclerosis_2NbHMSP; Human Fe
tal Lung III; T Cell helper I was found to be expressed.

Preferred epitopes include those comprising the sequence shown in SEQ ID NO: 82 as residues Ser-12 to Cys-40.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 34 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 612 of SEQ ID NO: 34, and b is 15 to 6
26, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 34, and wherein b is a + 14 or greater), excluding one or more polynucleotides You.

(Characteristics of Protein Encoded by Gene No. 25) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gi | 159655 (in which “ colagen [Ascaris suum])
(All information available through the cited accession numbers is incorporated herein by reference) and has determined to share sequence homology with the sequences available through.
A partial alignment demonstrating the observed homology is shown immediately below.

[0133]

Embedded image The gi | 159655 segment shown above as "Sbjct" is shown as the sequence of SEQ ID NO: 143. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art, some of which 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 present invention can be queried in the alignments shown above (gaps introduced into the sequence by computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 144 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Human Osteo
sarcoma; Meningima, M1; Human Fetal D
ura Mater; Spinal cord; Human Synovial
It was found to be predominantly expressed in Sarcoma.

Preferred epitopes include residues Asn-40-Ala-55, Pro-7
3- to Arg-89 and Arg-110 to Gly-116 include epitopes containing the sequence shown in SEQ ID NO: 83.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 35 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1816 of SEQ ID NO: 35, and b is 15 to
An integer of 1830, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 35, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of the Protein Encoded by Gene No. 26) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gi | 2621329 (as a non-limiting example)
Among them, "ferredoxin [Methanobacterium the
(rmoautotropicum) ”(all information available through the accession number cited is hereby incorporated by reference) and has determined to share sequence homology with the sequences available through this publication. A partial alignment demonstrating the observed homology is shown immediately below.

[0139]

Embedded image The segment of gi | 2621329, shown above as "Sbjct",
This is shown as the sequence of SEQ ID NO: 145.

Preferred polypeptides of the present invention can be queried in the alignment shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 146 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Soares infa
nt brain 1NIB; mainly expressed in Human Cerebellum and, to a lesser extent, Human endometrial
somales adult brain N2b
5HB55Y; Human Umbilical Vein Endothel
ial cells, frac B, re-excision; Soares_
fetal_heart_NbHH19W; Soares_sensent
_Fibroblasts_NbHSF; Salary Grand, Li
b3; Amniotic Cells-Primary Culture; w
ilm's tumor; Human Fetal Dura Mater; H
human Fetal Brain; Soares breast 2NbHB
st; Human Whole Six Week Old Embryo; A
dipocytes; Human Microvascular Endoth
eliial Cells, fracture. A: Monocyte activate
It was found to be expressed in ed.

[0142] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 36 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1927 of SEQ ID NO: 36, and b is 15 to
An integer of 1941, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 36, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of the Protein Encoded by Gene No. 27) Using the computer algorithm BLASTX, the translation product of this gene has the following database entry number gi | 146856 (
Among them, "glutamine-rich protein [Caenorha
bditis elegans]) (all information available through the accession number cited is hereby incorporated by reference) and has determined to share sequence homology with the sequences available through it. . A partial alignment demonstrating the observed homology is shown immediately below.

[0144]

Embedded image The segment of gi | 1465856, shown above as “Sbjct”,
This is shown as SEQ ID NO: 147.

[0145] Preferred polypeptides of the present invention can be queried in the alignment shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence set forth as SEQ ID NO: 148 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Soares adul
t brain Predominantly expressed in N2b4HB55Y and, to a lesser extent, Soares_fetal_lung_NbHL19W; Strat
antigen neuroepithelium NT2RAMI 937234
Soares_NhHMPU_S1; Soares infant bra
in 1NIB; cerebellum, Enzyme subtract
ed; Synovial hypoxia-RSF subtracted; H
human Man Depression Tissue; Human F
et al. was found to be expressed in Dura Mater.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 37 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1063 of SEQ ID NO: 37, and b is 15 to
An integer of 1077, where both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 37, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 28) This gene was found to be mainly expressed in Human Fetal Dura Mater.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 38 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1295 of SEQ ID NO: 38, and b is 15 to
An integer of 1309, where both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 38, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 29) This gene was found to be mainly expressed in Human Fetal Dura Mater.

[0151] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 39 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 896 of SEQ ID NO: 39, and b is 15 to 9
An integer of 10 wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 39, and wherein b is a + 14 or greater, excluding one or more polynucleotides You.

(Characteristics of the Protein Encoded by Gene No. 30) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database accession number gi | 2429380 (as a non-limiting example)
Among them, "C. elegans protein showering simil"
arity to a short region of the murin
e Cux homedomin protein ")
All information available through the cited accession numbers has been determined to share sequence homology with the sequences available through (incorporated herein by reference). A partial alignment demonstrating the observed homology is shown immediately below.

[0153]

Embedded image The segment of gi | 2429380, shown above as "Sbjct", is
This is shown as SEQ ID NO: 149.

Preferred polypeptides of the present invention can be queried in the alignment shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 150 corresponding to the sequence.

The gene encoding this disclosed cDNA is believed to be on chromosome 21. Therefore, the polynucleotide according to the present invention is useful as a marker in linkage analysis for chromosome 21.

This gene was obtained from the following tissue / cDNA library: Soares_preg
nant_uterus_NbHPU; Human Osteocluster
a, re-excision; Soares fetal river spl
een 1NFLS, and to a lesser extent Hum
an Fetal Spleen; Human Hypothalamus, s
chizophrenia, re-excision; Soares_NhHM
Pu_S1; Stratagene colon (# 937204);
mal cell TF274; Stratagene lung (# 9372
10); NCI_CGAP_Lu5; NCI_CGAP_GCB1; Colon
Carcinoma; found to be expressed in Soares infant brain 1NIB.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 40 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1915 of SEQ ID NO: 40, and b is 15 to
1929, where both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 40, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of the Protein Encoded by Gene No. 31) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database accession number gi | 3955310 (as a non-limiting example)
The sequence homology with the sequence available through it (described as "collapsin-2-like protein") (all information available through the cited accession number is hereby incorporated by reference). Decided to share. A partial alignment demonstrating the observed homology is shown immediately below.

[0159]

Embedded image The segment of gi | 3955310, shown above as "Sbjct",
It is shown as SEQ ID NO: 151 and / or SEQ ID NO: 153.

[0160] Preferred polypeptides of the present invention can be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence set forth as SEQ ID NO: 152 and / or SEQ ID NO: 154 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Stratagene
ovarian canceler (# 937219); Human Fetal
Spleen; Human 8 Week Whole Embryo was found to be mainly expressed.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 41 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1430 of SEQ ID NO: 41, and b is 15 to
An integer of 1444, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 41, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of the protein encoded by Gene No. 32) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database accession number gi | 488328 (in which " rabbit protein [Oryctolagus cnic]
ulus]) (all information available through the accession number cited is hereby incorporated by reference herein) and shares sequence homology with that available. Partial alignments demonstrating the observed homology are:
Shown directly below.

[0164]

Embedded image The gi | 488328 segment shown above as "Sbjct" is shown as SEQ ID NO: 155.

[0165] Preferred polypeptides of the present invention can be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 156 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Monocyte ac
activated, re-excision; Human Fetal Dura
It was found to be mainly expressed in Mater.

Tissue distribution in immune cells (eg, activated monocytes) indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosing and treating various immune system disorders. Representative uses are described in Examples 11, 13, 14, 16, 1
8, 19, 20, and 27 and elsewhere herein in "Immunological activity" and "Infectious diseases". Briefly, this expression indicates a role in regulating the growth, survival, differentiation, and / or activation of hematopoietic cell lines, including blood stem cells. Involvement in the regulation of cytokine production, antigen presentation, or other processes suggests utility for treating 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 affects arthritis, asthma,
Immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, chronic granulomatosis, inflammatory bowel disease, sepsis, contusion, neutropenia, neutrophilia, psoriasis, hypersensitivity (eg, T cell mediated Cell injury); immune response 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, drug-induced hemolysis) Anaemia, rheumatoid arthritis, Sjogren's disease, and scleroderma). In addition, the protein may represent a secreted factor that affects the differentiation or properties of other blood cells or recruits hematopoietic cells to the site of injury. Accordingly, this gene product is believed to be useful in the expansion of stem cells and related progenitor cells of various blood lineages, and in the differentiation and / or expansion of various cell types. In addition, the protein may also be used to determine biological activity, elicit antibodies (as tissue markers), isolate relevant ligands or receptors, identify agents that modulate their interactions, and Can be used as a nutritional supplement. The proteins, as well as antibodies against the proteins, may exhibit utility as tumor markers and / or immunotherapeutic targets for the tissues listed above.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 42 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1305 of SEQ ID NO: 42, and b is 15 to
An integer of 1319, where both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 42, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 33) This gene was obtained from the following tissue / cDNA library: Macrophage (
GM-CSF treated); Soares placenta Nb2H
P, and to a lesser extent Human Fetal
Lung; CD34 + cell, I; Aorta endothial
cells + TNF-a; human corpus colosum;
Jurkat T-cell G1 phase; Human Fetal D
ura Mater; Human Rhabdomyosarcoma; Bon
e Marrow Stromal Cell, untreated; Huma
n Liver, normal; Smooth muscle, serum i
nduced, re-exc; Macrophage-oxLDL, re-ex
Ction; Smooth mouse, serum treated; H
Macrophage (GM-CSF treated), re-excis
ion; Human Tests, Reexcision; Human Pl
acenta; CD34 positive cells (Cord Blood)
d); Hodgkin's Lymphoma II; Human Cereb
ellum was found to be expressed.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 43 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 779 of SEQ ID NO: 43, and b is 15 to 7
93, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 43, and wherein b is a + 14 or greater, excluding one or more polynucleotides You.

(Characteristics of the protein encoded by Gene No. 34) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gi | 403460 (in which " transformation-related protein [H
omo sapiens] (all information available through the accession number cited is hereby incorporated by reference) and has determined to share sequence homology with the sequences available through it. . A partial alignment demonstrating the observed homology is shown immediately below.

[0172]

Embedded image The gi | 403460 segment shown above as “Sbjct” is shown as the sequence of SEQ ID NO: 157.

Preferred polypeptides of the present invention can be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 158 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Human Fetal
Dura Mater; Soares infant brain 1NIB
And, to a lesser extent, Human Thymus
Human Fetal Lung III; Soares plant
a Nb2HP: Human Old Ovary; Early Stage
Human Lung, subtracted; Soares retina
N2b4HR; Human Lung Cancer, re-excision
Human Hypothalamus, schizophrenia, re
-Excision; NTERA2 + retinoic acid, 14
days; Jurkat T-Cell, S phase; Human Inf
ant Brain: H. Kidney Medulla, re-excisi
on; NCI_CGAP_GCB1; NCI_CGAP_Pr22; Human
Ovarian Cancer Reexcision; Soares ad
ult brain N2b5HB55Y; ULCERIVE COLI
is: Clontech human aorta polyA + mRNA (
# 6572): Human fetal heart, Lambda ZAP
Express; Soares_fetal_lung_NbHL19W; Hu
man Testes Tumor, re-excision; Pancrea
s Islet Cell Tumor; Human Fetal Heart
Endothelial-induced; Endothelial cel;
ls-control; Analog T-cell; Human Oste
oclastoma; CD34 positive cells (Cord B
human) Cell B Lymphoma; Osteobla
sts; was found to be expressed in Soares fetal river spring 1NFLS.

The present invention is further directed to fragments of the polynucleotide sequences described herein. For example, a polynucleotide sequence of the deposited cDNA or a fragment of the nucleotide sequence set forth in SEQ ID NO: 44 is at least about 15 nucleotides, and more preferably at least about 20 nucleotides, at least about 25 nucleotides, and even more preferably at least about 30 nucleotides. Nucleotides, at least about 35 nucleotides, and even more preferably at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides long, at least about 1
00 nucleotides long, at least about 125 nucleotides long, at least about 150 nucleotides long
Contemplates polynucleotide fragments at least 175 nucleotides in length, which are useful as diagnostic probes and primers as discussed herein. Of course, longer fragments of 200-1500 nucleotides in length may also, but not all, have been deposited with the deposited cDNA or SEQ ID NO: 44.
Are useful according to the invention, as are fragments corresponding to most of the nucleotide sequences as shown in For example, a fragment that is at least 20 nucleotides in length is the nucleotide sequence of the deposited cDNA or SEQ ID NO: 44.
Fragments containing 20 or more contiguous bases from the nucleotide sequence as shown in are contemplated. In this context, “about” refers to the specifically recited ranges, at either or both termini, larger or smaller by some (5, 4, 3, 2, 2, or 1) nucleotides Including range. Representative examples of polynucleotide fragments of the present invention include, for example, about 1 to about 50, about 51 to about 100, about 101 to about 150, about 151 to about 200, about 201 to about 250, about 251 to about 300, about 301 to about 350, about 3
51 to about 400, about 401 to about 450, about 451 to about 500, about 501 to about 55
0, about 551 to about 600, about 601 to about 650, about 651 to about 700, about 701
About 750, about 751 to about 800, about 801 to about 850, about 851 to about 900,
About 901 to about 950, about 951 to about 1000, about 1001 to about 1050, about 10
51 to about 1100, about 1101 to about 1150, about 1151 to about 1200, about 12
01 to about 1250, about 1251 to about 1300, about 1301 to about 1350, about 13
51 to about 1400, about 1401 to about 1450, about 1451 to about 1500, and 1501 to 1550, 551 to 1600, 1601 to 1650, 1651 to 1
700, 1701-1750, 1751-1800, 1801-1850, 18
51-1900, 1901-1950, 1951-2014 or a complementary strand thereof, or a fragment containing or consisting of cDNA contained in the deposited clone. In this context, "about" may include some of the specifically recited ranges, either or both termini,
(5, 4, 3, 2, or 1) nucleotides. In a further embodiment, a polynucleotide of the invention encodes a functional property of the corresponding protein.

One further embodiment of the present invention is a polypeptide comprising or consisting of one or more of the following polypeptide fragments of the polypeptide set forth in SEQ ID NO: 92:

[0177]

Embedded image Further, a further general embodiment of the invention provides a polypeptide fragment of the polypeptide shown in SEQ ID NO: 92 that exhibits biological activity or a fragment encoded by a deposited clone of the invention (eg, a GAS assay). Fragments that stimulate activation and / or proliferation of CD34 + cells).

When tested against the U937 bone marrow cell line, supernatants from cells containing this gene activated the GAS assay. Therefore, this gene is designated as Jak-S
It appears to activate myeloid cells via the TAT 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 signaling pathway involved in cell differentiation and proliferation. Thus, activation of the Jak-STAT pathway as reflected by GAS binding.

The polynucleotides and / or polypeptides of the invention and / or agonists and / or antagonists thereof may also be used for the expansion of immature hematopoietic progenitor cells (eg, granulocytes, macrophages or monocytes) (transiently). By specifically preventing their differentiation). These bone marrow cells can be cultured in vitro. Thus, the polynucleotides and / or polypeptides of the invention, or agonists or antagonists thereof, may be useful as modulators of hematopoietic stem cells in vitro for bone marrow transplantation and / or gene therapy purposes. Since stem cells are rare and most useful for transferring genes for gene therapy, the polynucleotides and / or polypeptides of the invention can be used to isolate an enriched population of stem cells. Stem cells contain cytotoxins (eg, 5-Fu).
) Can be enriched by culturing the cells in the presence of This cytotoxin readily kills dividing cells, where stem cells are protected by the polynucleotides and / or polypeptides of the invention. These stem cells can be returned to a bone marrow transplant patient or can then be used for transfection of the desired gene for gene therapy. In addition, the gene can be injected into an animal, resulting in the release of stem cells from the animal's bone marrow into peripheral blood. These stem cells can be isolated for the purpose of autologous bone marrow transplantation or for manipulation of gene therapy. After the patient has completed chemotherapy or radiation treatment, the isolated stem cells can be returned to the patient.

[0180] Polynucleotides and polypeptides corresponding to this gene are also useful in the treatment and diagnosis of hematopoietic-related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia, or leukemia. This is because stromal cells are important in the production of hematopoietic lineage cells. Its uses include bone marrow cell ex vivo culture, bone marrow transplantation, bone marrow remodeling, neoplastic radiotherapy or chemotherapy.

The gene product may also be involved in regulating cytokine production, antigen presentation, or other processes, suggesting utility in treating cancer (eg, by boosting an immune response). This gene is expressed in cells of lymphoid origin, indicating that this natural gene product is involved in immune function.
Thus, it also includes arthritis, asthma, immunodeficiency disorders such as AIDS, leukemia,
Rheumatoid arthritis, chronic granulomatosis, inflammatory bowel disease, sepsis, contusion, neutropenia, neutropenia, psoriasis, hypersensitivity (eg, T-cell mediated cytotoxicity); immunity to transplanted organs and tissues Responses (eg, host versus graft and graft versus host disease) or autoimmune disorders (eg, autoimmune infertility, lens tissue injury, demyelination, systemic lupus erythematosus, drug-induced hemolytic anemia, rheumatoid arthritis, Sjogren Disease, and scleroderma) can be useful as agents for immunological disorders.

In addition, this protein may represent a secreted factor that affects the differentiation or properties of other blood cells or recruits hematopoietic cells to the site of injury. Thus, this gene product is used in the growth of stem cells and related progenitor cells of various blood lineages.
And may be useful in the differentiation and / or proliferation of various cell types. In addition, the protein may also be used to determine biological activity, elicit antibodies (as tissue markers), isolate relevant ligands or receptors, identify agents that modulate their interactions, and Can be used as a nutritional supplement. The proteins, as well as antibodies against the proteins, may exhibit utility as tumor markers and / or immunotherapeutic targets for the tissues listed above.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 44 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 2000 of SEQ ID NO: 44, and b is 15 to
2014, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 44, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of the Protein Encoded by Gene No. 35) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gi | 4102039 (as a non-limiting example)
Among them, "(AF008196) bax epsilon [Homo sapi
ens] ”(all information available through the cited accession numbers is incorporated herein by reference) and has determined to share sequence homology with the sequences available through. A partial alignment demonstrating the observed homology is shown immediately below.

[0185]

Embedded image The segment of gi | 4102039, shown above as "Sbjct",
This is shown as SEQ ID NO: 159. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art, some of which are described elsewhere herein. Assays to determine such activity are also
It is known in the art, some of which are described elsewhere herein.

Preferred polypeptides of the present invention can be queried in the alignment shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 160 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Human Fetal
Dura Mater; breast lymph node CDNA l
ibrary; found to be mainly expressed in Osteoblasts.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 45 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1186 of SEQ ID NO: 45, and b is 15 to
An integer of 1200, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 45, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of the protein encoded by Gene No. 36) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gi | 288145 (in which " human protein encoded by a putat
(described as "live ORF") (all information available through the cited accession number is incorporated herein by reference) and has determined to share sequence homology with the sequence available. A partial alignment demonstrating the observed homology is shown immediately below.

[0190]

Embedded image The gi | 288145 segment shown above as "Sbjct" is shown as SEQ ID NO: 161.

Preferred polypeptides of the present invention can be queried in the alignment shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 162 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Suppt cells,
cyclohexamide treated, differentially
expressed; H Female Bladerder, Adult; Fe
tal Liver, subtraction II; Human Heart
Human Thymus; Soares_pregnant_uterus
_NbHPU; Hemangiopericity toma; Colon Tumo
r; Resting T-Cell Library, II; Adipocyt
es; human tonsils; was found to be mainly expressed in Keratinocyte.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 46 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 1222 of SEQ ID NO: 46, and b is 15 to
An integer of 1236, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 46, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 37) This gene was obtained from the following tissue / cDNA library: Fetal River
, Subtraction II; was found to be predominantly expressed in Bone marrow.

[0195] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 47 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1281 of SEQ ID NO: 47, and b is 15 to
An integer of 1295, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 47, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 38) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database accession number gi | 2072792 (as a non-limiting example)
Among them, "matrilin-2 precursor [Mus muscul
us] ") (all information available through the cited accession numbers is hereby incorporated by reference) and has determined to share sequence homology with the sequences available through. A partial alignment demonstrating the observed homology is shown immediately below.

[0197]

Embedded image The segment of gi | 2072792, shown above as "Sbjct",
SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169 and / or SEQ ID NO: 171. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art, some of which 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 present invention can be queried in the alignment shown above (gaps introduced into the sequence by computer are of course removed).
SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 1 corresponding to the sequence
70 and / or a polypeptide having the amino acid sequence set forth as SEQ ID NO: 172.

This gene was obtained from the following tissue / cDNA library: Soares feta
predominantly expressed in I liver spring 1NFLS, and to a lesser extent,

[0200]

Embedded image Was found to be expressed in

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 48 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 2149 of SEQ ID NO: 48, and b is 15 to
An integer of 2163, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 48, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 39) This gene was found to be mainly expressed in Fetal Liver, subtraction II.

[0203] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 49 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1219 of SEQ ID NO: 49, and b is 15 to
An integer of 1233, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 49, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 40) Using the computer algorithm BLASTX, the translation product of this gene may have the following database entry number dbj || AB0076 as a non-limiting example:
38_14 (in which "(AB007638) function unknown"
n [Bacillus subtilis]) (all information available through the cited registration number is hereby incorporated by reference).
Determined to share sequence homology with the sequences available through. A partial alignment demonstrating the observed homology is shown immediately below.

[0205]

Embedded image The segment of dbj | AB007638_14, shown above as "Sbjct", is shown as the sequence of SEQ ID NO: 173.

[0206] Preferred polypeptides of the present invention can be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 174 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Soares adul
t brain is predominantly expressed in N2b4HB55Y and, to a lesser extent,

[0208]

Embedded image Was found to be expressed in

[0209] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 50 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1767 of SEQ ID NO: 50, and b is 15 to
1781, where both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 50, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 41) This gene was found to be mainly expressed in Human Fetal Dura Mater.

[0211] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 51 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 and 823 of SEQ ID NO: 51, and b is 15 to 8
37, where both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 51, and where b is a + 14 or greater), excluding one or more polynucleotides You.

(Characteristics of the Protein Encoded by Gene No. 42) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gi | 288145 (in which " human protein encoded by a putat
(described as "live ORF") (all information available through the cited accession number is incorporated herein by reference) and has determined to share sequence homology with the sequence available. A partial alignment demonstrating the observed homology is shown immediately below.

[0213]

Embedded image The gi | 288145 segment shown above as "Sbjct" is shown as the sequence of SEQ ID NO: 175.

[0214] Preferred polypeptides of the present invention can be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 176 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Human Fetal
It is mainly expressed in Lung III, and to a lesser extent Hum
an Adult Retina; Early Stage Human Lu
ng, subtracted; KMH2; Human Fetal Dura
Mater; Liver, Hepatoma; Primary Dendrit
It was found to be expressed in ic Cells, lib1.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 52 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1363 of SEQ ID NO: 52, and b is 15 to
An integer of 1377, where both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 52, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 43) This gene was obtained from the following tissue / cDNA library: Human T-cel
l lymphoma, re-excision; Colon Carcino
ma; Brain frontal cortex; Nine Week Ol
d It was found to be predominantly expressed in Early Stage Human.

[0219] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 53 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1815 of SEQ ID NO: 53, and b is 15 to
An integer of 1829, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 53, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 44) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gi | 108544 (
Among them, "metastasis-associated gene [Homo
sapiens] ”(all information available through the cited accession numbers is incorporated herein by reference) and has determined to share sequence homology with the sequences available through. A partial alignment demonstrating the observed homology is shown immediately below.

[0220]

Embedded image The segment of gi | 100544, shown above as “Sbjct”,
This is shown as SEQ ID NO: 177. Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity. Such activities are known in the art, some of which are described elsewhere herein. Assays to determine such activity are also
It is known in the art, some of which are described elsewhere herein.

Preferred polypeptides of the present invention can be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 178 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Soares_feta
It is predominantly expressed in 1_heart_NbHH19W and, to a lesser extent, Stratagene ovarian cancer (# 93721).
9); NCI_CGAP_GCB1; NCI_CGAP_Alv1; NCI_C
GAP_Pr12; Thymus; Human (Caco-2) cell li
ne, adenocarcinoma, colon, remake; Human
Primary Breast Cancer, re-excision; S
upCells, cyclohexamide treated; L428
; Human Jurkat Membrane Bound Polysom
es; Human Uterine Cancer; Pancreas Isl
et Cell Tumor; Human Placenta; Brain f
roman cortex; Human Testes; Bone Marr
ow Cell Line (RS4, 11); Stratagene colo
n (# 937204); Human 8 Week Whole Embryo
Was found to be expressed in

The tissue distribution (eg, testis tissue, endometrial tumors, ovarian cancer, adenocarcinoma) and the fact that this gene shares sequence homology with metastasis-related genes indicates that the polynucleotides and polymorphisms corresponding to this gene The peptides indicate that they are useful for diagnosing and / or treating germline cancers whose expression is observed, and cancers of other tissues. further,
This protein can also be used to determine biological activity, to raise antibodies (
(As tissue markers), to isolate related ligands or receptors, to identify agents that modulate their interaction, and as a nutritional supplement.
The proteins, as well as antibodies against the proteins, may exhibit utility as tumor markers and / or immunotherapeutic targets for the tissues listed above.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 54 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1567 of SEQ ID NO: 54, and b is 15 to
An integer of 1581, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 54, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of the protein encoded by Gene No. 45) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database accession number gi | 171216 (
It is described as "unknown [Homo sapiens]" therein.)
(All information available through the cited accession numbers is incorporated herein by reference) and has determined to share sequence homology with the sequences available through.
A partial alignment demonstrating the observed homology is shown immediately below.

[0226]

Embedded image The segment of gi | 171216, shown above as “Sbjct”,
It is shown as a sequence of SEQ ID NO: 179 and / or SEQ ID NO: 181.

[0227] Preferred polypeptides of the present invention may be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 180 and / or SEQ ID NO: 182 corresponding to the sequence.

This gene was obtained from the following tissue / cDNA library: Monocyte ac
activated; Apoptotic T-cell; Brain front
It was found to be predominantly expressed in al cortex.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 55 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1506 of SEQ ID NO: 55, and b is 15 to
An integer of 1520, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 55, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 46) This gene was found to be mainly expressed in the Brain frontal cortex.

[0231] Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 56 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1249 of SEQ ID NO: 56, and b is 15 to
An integer of 1263, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 56, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of the protein encoded by Gene No. 47) Using the computer algorithm BLASTX, the translation product of this gene was identified by the following database entry number gi | 555375 (in which " envelope glycoprotein gp46 [Simia
n T-cell lymphotropic viral type 1] "
All information available through the cited accession numbers (as described herein) is determined to share sequence homology with the sequences available through. A partial alignment demonstrating the observed homology is shown immediately below.

[0233]

Embedded image The gi | 555375 segment shown above as "Sbjct" is shown as SEQ ID NO: 183.

[0234] Preferred polypeptides of the present invention can be queried in the alignments shown above (gaps introduced into the sequence by the computer are of course removed).
Includes a polypeptide having the amino acid sequence shown as SEQ ID NO: 184 corresponding to the sequence.

The gene was obtained from the following tissue / cDNA library: Brain front
al cortex; Human Amygdala; Soares_preg
nant_uterus_NbHPU; Human Testes; Human
8 Week Whole Embryo; Soares infant b
It was found to be predominantly expressed in the rain 1 NIB.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 57 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1333 of SEQ ID NO: 57, and b is 15 to
An integer of 1347, wherein both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 57, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

(Characteristics of Protein Encoded by Gene No. 48) This gene was obtained from the following tissue / cDNA library: Human Whole
Brain # 2-Oligo dT> 1.5 Kb;
It was found to be mainly expressed in ntal cortex.

Many polynucleotide sequences (eg, EST sequences) are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 58 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing all related sequences is cumbersome. Therefore,
Preferably, from the present invention, the nucleotide sequence described by the general formula ab (
Here, a is an arbitrary integer between 1 to 1263 of SEQ ID NO: 58, and b is 15 to
An integer of 1277, where both a and b correspond to nucleotide residue positions set forth in SEQ ID NO: 58, and where b is a + 14 or greater.
One or more polynucleotides are excluded.

[0239]

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

The cDNA clone ID was deposited on that date and is referred to as “ATCC Accession No. Z
And the corresponding accession numbers listed under "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.

“Total nucleotide sequence” refers to the total number of nucleotides in the contig identified by “Gene No.”. The deposited clone may contain all or most of these sequences, and these may be referred to as “5 of the cloned sequences” of SEQ ID NO: X.
It is reflected by nucleotide positions indicated as 'nucleotides' and '3' nucleotides of the clone sequence. SEQ ID for putative start codon (methionine)
The nucleotide position of ID NO: X is “5 ′ nucleotide of start codon”
Identified as Similarly, the nucleotide position of SEQ ID NO: X of the putative signal sequence is "5 'nucleotide of the first amino acid of the signal peptide."
Identified as

The translated amino acid sequence starts with methionine and begins with “amino acid SEQ ID
Although identified as "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 acid of SEQ ID NO: Y of the putative signal peptide are identified as “first amino acid of signal peptide” and “last amino acid of signal peptide”. The predicted first amino acid position in SEQ ID NO: Y for a secretory moiety is identified as "putative first amino acid for secretory moiety". Finally, the amino acid position of SEQ ID NO: Y of the last amino acid in the open reading frame is identified as "last amino acid of ORF".

SEQ ID NO: X (where X can be any of the polynucleotide sequences disclosed in the Sequence Listing) and translated SEQ ID NO: Y (where Y is disclosed in the Sequence Listing) (Which can be any polypeptide sequence) is sufficiently accurate and is otherwise suitable for various uses well known in the art and those further described below. For example, SEQ ID NO: X is
Useful for designing nucleic acid hybridization probes that detect the nucleic acid sequence contained in SEQ ID NO: X or the cDNA contained in the deposited clone. These probes also hybridize to nucleic acid molecules in a biological sample, thereby enabling various forensic and diagnostic methods of the present invention. Similarly, the polypeptide identified from SEQ ID NO: Y is
For example, it can be used to generate antibodies that specifically bind to the proteins (including polypeptides and secreted proteins) encoded by the cDNA clones identified in Table 1.

Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. This error, as a misidentified nucleotide,
Alternatively, it is present 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 putative amino acid sequence. In these cases, even though the DNA sequence produced may be more than 99.9% identical to the actual DNA sequence (eg, insertion or deletion of one base 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 requiring accuracy in nucleotide or amino acid sequence, the present invention relates to a generated nucleotide sequence identified as SEQ ID NO: X, and as SEQ ID NO: Y. The ATC as described in Table 1 as well as the putative translated amino acid sequence identified
Also provided is a sample of plasmid DNA comprising the human cDNA of the present invention deposited at C. 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 a deposit. 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, including the deposited human cDNA, collecting the protein, and encoding the sequence. By determining, it can be determined directly.

The present invention also relates to the gene corresponding to SEQ ID NO: X, SEQ ID NO: Y, or the deposited clone. The corresponding gene can be isolated according to known methods, using the sequence information disclosed herein. Such methods include preparing a probe or primer from the disclosed sequences, and identifying or amplifying the corresponding gene from a suitable source of genomic material.

Also provided in the present invention are allelic variants, orthologs (or
tholog), and / or species homologs. SEQ ID NO: X, SEQ ID NO: Y or the deposited clones may be constructed using procedures known in the art and using the sequences disclosed herein or information from the clones deposited with the ATCC. One may obtain full length genes, allelic variants, splice variants, full length coding portions, orthologs, and / or species homologs of the corresponding gene. For example, allelic variants and / or species homologs make appropriate probes or primers from the sequences provided herein, and provide an appropriate nucleic acid source for the allelic variant and / or desired homolog. It can be isolated and identified by screening.

[0249] 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 a combination of these methods. And the like. Means for preparing such polypeptides are well understood in the art.

The polypeptide may be in the form of a secreted protein (including the mature form) or may 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, prosequences, 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 preferably are substantially purified. Recombinantly produced versions of the polypeptides (including secreted polypeptides) can be obtained using the techniques described herein or other techniques known in the art (eg, Smith and John).
son, Gene 67: 31-40 (1988)). The polypeptides of the present invention also include
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 invention also provides a polypeptide comprising or consisting of a polypeptide sequence of SEQ ID NO: Y and / or a cDNA contained in ATCC deposit Z. SEQ ID
NO: Y polypeptide sequence and / or cD contained in ATCC deposit Z
Polynucleotides encoding a polypeptide comprising or consisting of a polypeptide sequence encoded by NA are also encompassed by the present invention.

Signal Sequences The invention also includes the mature forms of the polypeptides having the polypeptide sequence of SEQ ID NO: Y and / or the polypeptide sequence encoded by the cDNA in the deposited clone. Polynucleotides encoding the mature form (such as, for example, the polynucleotide sequence of SEQ ID NO: X and / or the polynucleotide sequence contained in the cDNA of the deposited clone) are also included in the invention. According to the signal hypothesis, once transport of growing protein chains across the rough endoplasmic reticulum is initiated, proteins secreted by mammalian cells are:
Has a signal sequence or secretory leader sequence that is cleaved from the mature protein. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of secreted proteins is completely
Not homogeneous, this results in more than one mature species of the protein. Furthermore, it has long been known that the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the entire protein (ie, it is unique to the amino acid sequence of this 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 the short N-terminal charged region followed by the uncharged region of the complete (uncleaved) protein. von Heinje, Nucleic Acids Res
. 14: 4683-4690 (1986) uses information from the residues surrounding the cleavage site (typically -13 to +2 residues), where +1 indicates the amino terminus of the secreted protein. . For each of these methods, the accuracy of predicting the breakpoint of a known mammalian secretory protein is in the range of 75-80% (
von Heinje, supra). However, the two methods do not always generate the same putative breakpoint for a given protein.

In the case of the present invention, the deduced amino acid sequence of the secreted polypeptide is Si
gnalP (Henrik Nielse)
n et al., Protein Engineering 10: 1-6 (1997)).
Was analyzed by This program predicts the location of a protein in a cell based on the amino acid sequence. As part of the computer prediction of this localization, Mc
The method of Geoch and von Heinje is incorporated. By this program, the analysis of the amino acid sequence of the secreted proteins described herein is shown in Table 1.
Provided the results shown in

However, as will be appreciated by those skilled in the art, the cleavage site will sometimes vary from organism to organism and cannot be predicted with absolute certainty. Therefore, the present invention provides
Provided is a secreted polypeptide having the sequence set forth in ID NO: Y, which has an N-terminus beginning within 5 residues (ie, + 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 entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Furthermore, the signal sequences identified by the above analysis cannot necessarily predict the naturally occurring signal sequence. For example, a naturally occurring signal sequence can be further upstream from a putative signal sequence. However, the putative signal sequence appears to be able to direct secreted proteins to the ER. Nevertheless, the present invention relates to the use of SEQ ID NOs in mammalian cells (eg, COS cells as described above).
: X provides a mature protein produced by expression of the polynucleotide sequence and / or the polynucleotide sequence contained in the cDNA of the deposited clone. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

(Polynucleotide and Polypeptide Variants) The present invention relates to modifications of the polynucleotide sequence disclosed in SEQ ID NO: X, the complementary strand thereto, and / or the cDNA sequence contained in the deposited clone. About the body.

The present invention also encompasses variants of the polypeptide sequences disclosed in SEQ ID NO: Y, and / or the polypeptide sequences encoded by the deposited clones.

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

The present invention also relates to, 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, SEQ ID NO: Y A nucleotide sequence encoding a polypeptide, a nucleotide sequence encoding a polypeptide encoded by a cDNA contained in a deposited clone, and / or a polynucleotide fragment of any of these nucleic acid molecules (e.g., as described herein) At least 80%, 85%, 90%, 95%
, 96%, 97%, 98%, or 99% identical nucleotide sequences. Polynucleotides that hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions, the polypeptides encoded by these polynucleotides, are also encompassed by the invention.

The present invention also provides a polypeptide sequence as set forth in, eg, SEQ ID NO: Y, a polypeptide sequence encoded by a cDNA contained in a deposited clone, and / or any of these polypeptides. For polypeptide fragments (eg, fragments described herein), at least 80
%, 85%, 90%, 95%, 96%, 97%, 98%, 99%.

By a nucleic acid having a nucleotide sequence that is at least 95% “identical” to a reference nucleotide sequence of the present invention, the nucleotide sequence of the nucleic acid may vary from 100 nucleotides of each of the reference nucleotide sequences whose nucleotide sequence encodes a polypeptide. It is intended to be identical to the reference sequence except that it can contain up to 5 point mutations per sequence. In other words, at least 9 relative to the reference nucleotide sequence.
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, as many as 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 identified as described herein.

In practice, any particular nucleic acid molecule or polypeptide will have at least 80%, 85%, 90%, 95%, 96%, 9%,
Whether it is 7%, 98%, or 99% identical can be determined conventionally using known computer programs. A preferred method for determining the best overall match (also referred to as an overall sequence alignment) between a query sequence (a sequence of the invention) and a subject sequence is described in Brutlag et al. (Comp. App
. Biosci. 6: 237-245 (1990)). In sequence alignment, the query and subject sequences are both DNA sequences. RNA
Sequences can be compared by converting U to T. The result of this global sequence alignment is expressed as 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, Randomize
ation Group Length = 0, Cutoff Score = 1,
Gap Penalty = 5, Gap Size Penalty 0.05,
Window Size = 500 or the length of the nucleotide sequence of interest (whichever is shorter).

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

For example, a 90 base subject sequence is aligned with a 100 base query sequence to determine percent identity. The deletion occurs at the 5 'end of the subject sequence, so the FASTDB alignment does not show a match / alignment at the first 10 bases at the 5' end. 1
Zero unpaired bases represent 10% of the sequence (number of bases at the unmatched 5 'and 3' ends / total number of bases in the query sequence), so that 10% is FASTD
It is subtracted from the percent identity score calculated by the B program.
If the remaining 90 bases match exactly, 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 which 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 'and 3' bases of the subject sequence that do not match / align with the query sequence are manually corrected. No other manual corrections are made for the purposes of the present invention.

An amino acid sequence of a subject polypeptide may differ from the query amino acid sequence of the invention by, for example, a polypeptide having an amino acid sequence that is at least 95% “identical” to the subject amino acid sequence. It is intended to be identical to the query sequence except that it can include up to 5 amino acid changes per 100 amino acids. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence, up to 5% of the amino acid residues in the subject sequence will have insertions, deletions, (indels)
Or it can be substituted with another amino acid. These modifications of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence, or may occur anywhere between those terminal positions, individually between residues in the reference sequence, or Are interspersed in any one or more of the following contiguous groups:

As a practical matter, any particular polypeptide may be selected, for example, from the amino acid sequence shown in Table 1 (SEQ ID NO: Y) or the amino acid encoded by the cDNA contained in the deposited clone. At least 80% of the sequence
, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or not, can be conventionally determined using known computer programs. The best overall match between the query sequence (sequence of the invention) and the subject sequence (
A 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 based on the algorithm. In a sequence alignment, the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The results of the overall sequence alignment described above are expressed as percent identity. FASTD
Preferred parameters used for the B amino acid alignment are: Matrix = PAM
0, k-tuple = 2, Mismatch Penalty = 1, Joini
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 amino acid sequence of interest (whichever is shorter).

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

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence, and thus the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. Ten unpaired residues represent 10% of the sequence (non-matching N-terminal and C
Number of residues at the end / total number of residues in the query sequence).
10% is subtracted from the percent identity score calculated by the DB program. If the remaining 90 residues match exactly, 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- 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 the residue positions outside the N- and C-termini of the subject sequence that do not match / align with the query sequence, as indicated in the FASTDB alignment, are manually corrected.
No other manual corrections are made for the purposes of the present invention.

Variants can include changes in the coding region, the non-coding region, or both. Particularly preferred are those that 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 generated by silent substitutions due to the degeneracy of the genetic code are preferred. In addition, 5 in any combination
Variants in which -10, 1-5, or 1-2 amino acids are substituted, deleted, or added are also preferred. Polynucleotide variants may optimize codon expression for a particular host (e.g., codons in human mRNA may be E. coli) for a variety of reasons.
to a preferred codon by a bacterial host such as E. coli)))
Can be generated.

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

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

Furthermore, a wealth of 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 an 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 "most of the molecules can be changed with little effect on either [binding activity or biological activity]." (See summary). In fact, of more than 3,500 nucleotide sequences tested,
As few as 23 unique amino acid sequences produced proteins that differed significantly in activity from the wild type.

In addition, deletion of one or more amino acids from the N-terminus or C-terminus of a polypeptide may result in alteration or deletion of one or more biological functions, but may not affect other biological functions. Activity may still be retained. For example, the ability of a deletion variant to induce and / or bind an antibody that recognizes a secreted form is determined when fewer than the majority of residues in the secreted form are removed from the N-terminus or C-terminus. Seems to be retained. Whether a particular polypeptide lacking the N- or C-terminal residue of a protein retains such immunogenic activity depends on the conventional methods described herein, and If not, it can be easily determined by a conventional method known in the art.

Thus, the present invention further includes polypeptide variants that exhibit substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions that are selected according to general rules known in the art, with 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 major 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 where 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 modified, but still maintain the biological activity of the protein.

A 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 (
Introducing one alanine mutation at each residue in the molecule) can be used. (Cunni
ngham 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 mostly buried (within the tertiary structure of the protein) require non-polar side chains, while the characteristics of surface side chains are generally poorly preserved. Further, conservative amino acid substitutions that are tolerated include substitution of Ala, Val, Leu, and Ile for aliphatic or hydrophobic amino acids; substitution of Ser and Thr for hydroxyl residues; substitution of Asp and Glu for acidic residues; Asn of amide residue
And Gln substitutions, basic residue Lys, Arg, and His substitutions; aromatic residue Phe, Tyr, and Trp substitutions, and small-sized amino acids Ala, Ser, Thr, Met, and Gly. Including substitutions.

In addition to conservative amino acid substitutions, the variants of the present invention may include (i) substitutions with one or more non-conservative amino acid residues, wherein the substituted amino acid residue is An encoded amino acid residue or not, or (ii) 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) Fc fusion region peptide, or a leader or secretory sequence, or a sequence that facilitates purification). Such variant polypeptides are considered to be within the purview of those skilled in the art given the teachings herein.

For example, a polypeptide variant that includes an amino acid substitution of a charged amino acid with another charged or neutral amino acid may result in proteins with improved properties (eg, less aggregation). Can be generated. Aggregation of a pharmaceutical formulation results in both reduced activity and increased clearance due to the immunogenic activity of the aggregate. (Pinkard 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)).

A further embodiment of the present invention comprises at least one amino acid substitution, wherein
Amino acids of the invention having an amino acid sequence that does not exceed an amino acid substitution, even more preferably does not exceed a 40 amino acid substitution, even more preferably does not exceed a 30 amino acid substitution, and even more preferably does not exceed a 20 amino acid substitution. It relates to a polypeptide comprising the sequence. Of course, in order of increasing preference, the peptide or polypeptide will contain at least one amino acid substitution, but not more than 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, the mature forms and / or other fragments described herein) is from 1 to 5, 5-10, 5-25, 5-50, 10-50, or 50
~ 150, with conservative amino acid substitutions preferred.

(Polynucleotides and Polypeptide Fragments) The present invention also relates to polynucleotide fragments of the polynucleotide of the present invention.

In the present invention, a “polynucleotide fragment” refers to a short polynucleotide having the following nucleic acid sequence: a polynucleotide contained in a deposited clone or encoded by a cDNA in a deposited clone. A part of the nucleic acid sequence encoding the peptide; a part of the nucleic acid sequence shown in SEQ ID NO: X or the complement thereof, or a part of the polynucleotide sequence encoding the polypeptide of SEQ ID NO: Y is there. The nucleotide fragment of the present invention is preferably 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 15 nt. It is 150 nt long. For example, a fragment "at least about 20 nt in length" is intended to include 20 or more contiguous bases from the cDNA sequence contained in the deposited clone or the nucleotide sequence set forth in SEQ ID NO: X.
In this context, "about" means specifically or specifically (at either or both ends, several (5, 4, 3, 2, or 1) nucleotides larger or smaller than this value. Value). These nucleotide fragments have uses, such as, but not limited to, as diagnostic probes and primers, as discussed herein. Of course, larger fragments (eg, 50, 150, 500, 600, 2000 nucleotides)
Is preferred.

Further, representative examples of the polynucleotide fragment of the present invention include, for example,
Fragments comprising the sequence of the following approximate number of nucleotides or consisting of: 1-50, 51-100, 101-150, 151-20
0, 201-250, 251-300, 301-350, 351-400, 40
1 to 450, 451 to 500, 501 to 550, 551 to 600, 651 to 70
0, 701 to 750, 751 to 800, 800 to 850, 851 to 900, 90
1 to 950, 951 to 1000, 1001 to 1050, 1051 to 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, 1551-1600, 1601-1650
1651-1700, 1701-1750, 1751-1800, 1801-
1850, 1851-1900, 1901-1950, 1951-2000, or the end of SEQ ID NO: X, or a complementary strand thereof, or cDNA contained in the deposited clone. In this context, "about"
The specifically recited ranges, and ranges at either or both termini, are greater or smaller by a few (5, 4, 3, 2, or 1) nucleotides. Preferably, these fragments encode a polypeptide having biological activity. More preferably, these polynucleotides may be used as probes or primers as discussed herein above. Polynucleotides that hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also included in the invention, as are the polypeptides encoded by these polynucleotides.

In the present invention, a “polypeptide fragment” refers to an amino acid sequence contained in SEQ ID NO: Y or a part of the amino acid sequence encoded by the cDNA contained in the deposited clone. A protein (polypeptide) fragment can be "free-standing" or within a larger polypeptide, where the fragment comprises a portion or region (most preferably as a single contiguous region). To form). Representative examples of polypeptide fragments of the present invention include, for example, fragments comprising or consisting of the following approximate amino acids: 1-20, 21-40, 41- 60, 61 to 80, 81 to 100, 10
2 to 120, 121 to 140, 141 to 160, or 161 to the end of the coding region. Further, the polypeptide fragments may be about 20, 30, 40, 50,
60, 70, 80, 90, 100, 110, 120, 130, 140, or 1
It can be 50 amino acids long. In this context, "about"
The specifically described 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 termini are included. 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 the carboxy terminus or both. For example, any number of amino acids (ranging from 1 to 60) can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids (ranging from 1 to 30)
It can be deleted from the carboxy terminus of the secreted protein or the mature form. further,
Any combination of the above amino and carboxy terminal deletions is preferred.
Similarly, polynucleotides encoding these polypeptide fragments are also preferred.

Structural or functional domains (eg, α-helix and α-helix forming regions, β-sheet and β-sheet forming regions, turns and turn forming regions, coils and coil forming regions, hydrophilic regions, Hydrophobic region, α amphiphilic region, β
Also preferred are fragments of polypeptides and polynucleotides characterized by an amphipathic region, a flexible region, a surface-forming region, a substrate binding region, and a fragment that includes a highly antigenic indicator region). Polypeptide fragments of SEQ ID NO: Y contained within a conserved domain are specifically contemplated by the present invention. In addition, polynucleotides encoding these domains are also contemplated.

[0287] 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 polypeptide of the present invention. The biological activity of the fragment may include an improved desired activity or a reduced undesirable activity. Polynucleotides encoding these polypeptide fragments also include
Included in the present invention.

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

[0291] The functional activity of the polypeptides of the present invention, and fragments, variants, derivatives, and analogs thereof, can be assayed by various methods.

For example, in one embodiment, various immunoassays known in the art to assay 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. Can be used. Such assays include radioimmunoassays, ELISAs (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoradiometric assays, gel diffusion sedimentation reactions, immunodiffusion assays, in situ immunoassays (e.g., colloidal gold, enzyme or Radioisotope labeling), Western blot, sedimentation reaction, agglutination assay (eg, gel agglutination assay, hemagglutination assay), complement binding assay,
Includes, but is not limited to, competitive and non-competitive assay systems using techniques such as immunofluorescence assays, protein A assays, and immunoelectrophoresis assays. 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 detecting binding in immunoassays and are within the scope of the invention.

In another embodiment, if the ligand for an identified polypeptide of the invention or the polypeptide fragment, variant, or derivative of the invention is evaluated for its ability to multimerize, binding may be, for example, Can be assayed by means well known in the art, such as reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting.
Generally, see Physicky, E .; Et al., 1995, Microbiol. Rev
. 59: 94-123. In another embodiment, the physiological correlation (signaling) of binding of the 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 invention and fragments, variants, derivatives, and analogs thereof. Can be routinely applied to determine the ability to elicit (either in vitro or in vivo) a relevant biological activity associated with the biological activity of a polypeptide of the invention. Other methods are known to those skilled in the art and are within the scope of the present invention.

Epitopes and Antibodies The present invention hybridizes to an epitope of a polypeptide having the amino acid sequence of SEQ ID NO: Y, or the polynucleotide sequence contained in ATCC accession number Z, or to the complement of the sequence of SEQ ID NO: X. By an epitope of the polypeptide sequence encoded by the polynucleotide or a polynucleotide sequence contained in ATCC Deposit No. Z under stringent hybridization conditions or lower stringency hybridization conditions as defined above. Includes polypeptides comprising or consisting of an epitope of the encoded polypeptide sequence. The present invention further provides 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 complementary to a polynucleotide sequence encoding an epitope of the present invention, And polynucleotide sequences that hybridize to complementary strands under stringent hybridization conditions or lower stringency hybridization conditions as defined above.

The term “epitope” as used herein refers to a portion of a polypeptide that has antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably a human. In a preferred embodiment, the invention includes a polypeptide comprising an epitope encoding the polypeptide as well as a polynucleotide. As used herein, an "immunogenic epitope" is defined as a portion of a protein that elicits an antibody response in an animal, and can be obtained by any method known in the art (e.g., for the production of antibodies described below). Method). (See, eg, Geysen et al., Proc. Natl. Acad. Sci. USA 81: 3.
998-4002 (1983)). As used herein,
The term "antigenic epitope" refers to a protein to which an antibody can immunospecifically bind to its antigen, as measured by any method known in the art (eg, the immunoassays described herein). Is defined as part of Immunospecific binding excludes non-specific binding, but need not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes do not necessarily require immunogenicity.

[0297] Fragments that function as epitopes can be generated by any conventional means. (See, for example, Houghten, Proc. Natl. Acad. Sci.
USA 82: 5131-5135 (1985) (U.S. Pat.
No. 1).

In the present invention, the antigenic epitope is preferably at least 4, at least 5, at least 6, at least 7, more preferably at least 8,
At least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and most Preferably it comprises a sequence of between about 15 and about 30 amino acids. Preferred polypeptides are at least 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100
Includes immunogenic or antigenic epitopes of amino acid residue length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, for raising antibodies (including monoclonal antibodies) that specifically bind to this epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as target molecules in immunoassays. (See, e.g., Wilson et al., Cell 37: 767-778 (19
84); Sutcliffe et al., Science 219: 660-666 (1
983)).

Similarly, immunogenic epitopes can be used to induce antibodies, for example, according to methods well known in the art. (See, eg, Sutcliffe et al., Supra); Wils.
on et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA
82: 910-914; and Bittle et al. 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 can be 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 to 10 amino acids
It has been shown to be sufficient to raise antibodies capable of (at least) binding to the linear epitope of the denatured polypeptide (eg, in Western blotting).

The epitope-bearing polypeptides of the present invention can be used to elicit 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.
See, eg, Sutcliffe et al., Supra; Wilson et al., Supra; and Bitt.
le et al. Gen. Virol. , 66: 2347-2354 (1985). When in vivo immunization is used, animals can be immunized with the free peptide; however, anti-peptide antibody titers can be measured using a macromolecular carrier (eg, keyhole limpet hemacyanin (KLH) or tetanus toxoid). Can be boosted. For example, a peptide containing a cysteine residue can be attached to the carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). On the other hand, other peptides can be conjugated to the carrier using more common binders such as glutaraldehyde. Animals, such as rabbits, rats, and mice, can be used with either free peptide or carrier-bound peptide, for example, in emulsion (about 100 μg of peptide or carrier protein and Freund's adjuvant or any known to stimulate an immune response). (Including other adjuvants) and / or intradermal injection. Several booster injections may be required, for example, at intervals of about two weeks to provide useful titers of the anti-peptide antibody. This titer can be detected, for example, by an ELISA assay using the free peptide adsorbed on the solid surface. The titer of the anti-peptide antibody in the serum from the immunized animal is increased by selection of the anti-peptide antibody (eg, by adsorption of the peptide on a solid support and elution of the selected antibody according to methods well known in the art). I can do it.

As will be appreciated by those of skill in the art, and as discussed above, polypeptides of the present invention that include an immunogenic or antigenic epitope can be fused to other polypeptide sequences. For example, a polypeptide of the invention may comprise an immunoglobulin (Ig
A, IgE, IgG, IgM) or their constants (CH1, C
H2, CH3, or any combination thereof and portions thereof), resulting in a chimeric polypeptide. Such a fusion protein may facilitate purification and increase in vivo half-life. This is shown for a chimeric protein consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of a mammalian immunoglobulin. See, for example, EP 394,827; Traunecker et al., Nature,
331: 84-86 (1988). Enhanced delivery of antigen across the epithelial disorder to the immune system has been demonstrated for antigen (eg, insulin) bound to an FcRn binding partner such as an IgG or Fc fragment (
See, for example, PCT publications WO 96/22024 and WO 99/04813). IgG fusion proteins having a disulfide bond dimer structure due to the disulfide bond of the IgG moiety are also more effective at binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone Was found. See, eg, Footoulakis et al. Biochem.
270: 3958-3964 (1995). Nucleic acids encoding the above-described epitopes can also be recombined with the gene of interest as an epitope tag (eg, a hemagglutinin ("HA") tag or a flag tag) to detect and purify the expressed polypeptide. Can help. For example, Janknecht
The system described by E. et al. Allows easy purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Na.
tl. Acad. Sci. ScL USA 88: 8972-897). In this system, the gene of interest is subcloned into a vaccinia recombinant plasmid, such that the open reading frame of the gene is translationally fused 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 the recombinant vaccinia virus are loaded onto Ni2 + nitriloacetic acid-agarose columns, and histidine-tagged proteins can be selectively eluted using imidazole-containing buffers.

Further fusion proteins of the invention can be produced 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 used to modulate the activity of the polypeptides of the invention, and using this method, polypeptides with altered activity, as well as agonists and antagonists of these polypeptides, can be produced. In general, U.S. Patent Nos. 5,605,793; 5,811,238;
Nos. 0,721; 5,834,252; and 5,837,458;
And Patten et al., Curr. Opinion Biotechnol.
8: 724-33 (1997); Harayama, Trends Biote.
chnol. 16 (2): 76-82 (1998); Hansson et al. M
ol. Biol. 287: 265-76 (1999); and Lorenzo.
And Blasco, Biotechniques 24 (2): 308-13.
(1998), each of which is hereby 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 achieved by DNA shuffling. D
NA shuffling involves assembling two or more DNA segments to produce a change 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) may be modified by subjecting them 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, etc.

(Antibodies) In addition, polypeptides of the present invention may be polypeptides, polypeptide fragments of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). Or a variant of SEQ ID NO: Y, and / or
Or an antibody that immunospecifically binds to an epitope and a T cell antigen receptor (T
CR). 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 anti-Id antibodies to the antibodies of the present invention), and any of the above-described epitope binding fragments. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that include an antigen binding site that immunospecifically binds to an antigen. Say. The immunoglobulin molecules of the present invention can be of any type (eg, IgG, IgE,
IgM, IgD, IgA, and IgY), any class (eg, IgG1,
IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass.

Most preferably, the antibody is a human antigen binding antibody fragment of the invention, including Fab, Fab ′ and F (ab ′) 2, Fd, single chain Fvs (sc
Fv), single chain antibodies, disulfide-linked Fvs (sdFv) and VL or V
Fragments that include any of the H domains include, but are not limited to. Antigen binding antibody fragments, including single chain antibodies, may include the variable region alone or in combination with all or part of the following: hinge region, CH1 domain, C1
H2 domain and CH3 domain. Also included in the invention are antigen binding fragments that also include any combination of a variable region with a hinge region, a CH1, a CH2 domain, and a CH3 domain. The antibodies of the invention can be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine (eg, mouse and rabbit), 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 Antibodies isolated from animals that do not express sex immunoglobulins (as described below and, for example, in US Pat. No. 5,939,598 to Kucherlapati et al.
As described in the item).

The antibodies of the present invention can be monospecific, bispecific, trispecific, or more multispecific. 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 solid support material). May be specific to For example, PCT publication WO 93/17715; WO 9/17715
WO08 / 00802; WO 91/00360; WO 92/05793; Tu
tt et al. Immunol. 147: 60-69 (1991); U.S. Pat.
No. 4,474,893, No. 4,714,681, No. 4,925,648,
Nos. 5,573,920 and 5,601,819; Kostelny et al.
J. Immunol. 148: 1547-1553 (1992).

The antibodies of the present invention can be described or specified in terms of the epitopes or portions of the polypeptides of the present invention to which they recognize or specifically bind. The portion of the epitope or polypeptide may be specified as described herein, for example, by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or as listed in the tables and figures. Can be Antibodies that specifically bind to any epitope or polypeptide of the present invention can also be excluded. Accordingly, the present invention includes antibodies that specifically bind a polypeptide of the present invention and allow for exclusion of a polypeptide of the present invention.

The antibodies of the present invention may 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 present 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 described herein) are also included in the invention. In certain embodiments, the antibodies of the invention cross-react with mouse, rat and / or rabbit homologs of human proteins and their corresponding epitopes. 9 for the polypeptide of the invention
Less than 5%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, 6
Binds polypeptides having an identity (as calculated using methods known in the art and described herein) of less than 5%, less than 60%, less than 55% and less than 50% Antibodies that do not are also included in the invention. In certain embodiments, the cross-reactivity is determined by any of the monospecific antigenic or immunogenic polypeptides disclosed herein, or 2, 3, 4, 5 or more specific It relates to a combination of antigenic and / or immunogenic polypeptides. Furthermore, antibodies that bind a polypeptide encoded by a polynucleotide that hybridizes to a polynucleotide of the invention under stringent hybridization conditions (as described herein) are included in the invention. . The antibodies of the present invention also include
They can be described or specified for their binding affinity to the polypeptides of the invention. Preferred binding affinities are less than 5 × 10 ⁻² M and 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, 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, 1
Less than 0 ^-7 M, less than 5 x 10 ^-8 M, less than 10 ^-8 M, less than 5 x 10 ^-9 M, less than 10 ^-9 M, less than 5 x 10 ^-10 M, less than 10 ^-10 M, 5 x Less than 10 ^-11 M, less than 10 ^-11 M,
5 × 10 ⁻¹² M or less, 10 ⁻¹² M or less, 5 × 10 ⁻¹³ M or less, 10 ⁻¹³ M or less, 5 ×
Affinities having a dissociation constant or Kd of less than 10 ^-14 M, less than 10 ^-14 M, less than 5 x 10 ^-15 M or less than 10 ^-15 M are mentioned.

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

The antibodies of the present invention may act as agonists or antagonists of the polypeptide of the present invention. For example, the invention includes antibodies that disrupt receptor / ligand interactions with the polypeptides of the invention, either partially or completely. Preferably, the antibodies of the invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention has the characteristics of both receptor-specific and ligand-specific antibodies. The invention also features receptor-specific antibodies that do not interfere with ligand binding but do interfere with receptor activation. Receptor activation (ie, signal transduction) can be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by phosphorylating the receptor (eg, tyrosine or serine / threonine), or detecting its substrate by immunoprecipitation followed by Western blot analysis (eg, as described above). . In certain embodiments, in the absence of the antibody, the ligand or receptor activity is increased by at least 95% of its 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 invention also relates to receptor-specific antibodies that prevent both ligand binding and receptor activation, as well as to recognize receptor-ligand complexes and, preferably, to bind unbound receptors or unbound ligands. It has the characteristics of a receptor-specific antibody that does not recognize E. coli. Similarly, the present invention relates to neutralizing antibodies that bind to a ligand and prevent the binding of the ligand to the receptor, and bind to the ligand, thereby preventing receptor activation but not preventing the ligand from binding the receptor. Including antibodies. Furthermore, the present invention includes antibodies that activate the receptor. These antibodies can act as receptor agonists, i.e., enhance or activate any or all of the biological activations of ligand-mediated receptor activation, e.g., by inducing receptor dimerization. obtain. The antibody can be specified as an agonist, antagonist or inverse agonist for a biological activity, including the specific biological activity of a peptide of the invention disclosed herein. The antibody agonist can be made using methods known in the art. See, for example, PCT Publication WO 96/40281; U.S. Patent 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. Immunol. 161 (4
): 1786-1794 (1998); Zhu et al., Cancer Res: 58.
(15): 3209-3214 (1998); Yoon et al. Immunol
. 160 (7): 3170-3179 (1998); Prat et al. Cell
. Sci. 111 (Pt2): 237-247 (1998); Pitard et al.
J. Immunol. Methods 205 (2): 177-190 (199
7); Liauard et al., Cytokine 9 (4): 233-241 (1
997); Carlson et al. 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, purify, detect and target a polypeptide of the invention. These include diagnostic and therapeutic methods both in vitro and in vivo. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of a polypeptide of the invention in a biological sample. For example, Harlo
W et al., Antibodies: A Laboratory Manual, (C
See Old Spring Harbor Laboratory Press, 2nd edition, 1988), which is hereby incorporated by reference in its entirety.

As discussed in further detail below, the antibodies of the present invention may be used either alone or in combination with other compounds. The antibody can be further recombinantly fused to the polypeptide or other compound at the N-terminus or C-terminus to a heterologous polypeptide, or chemically conjugated (including covalent and non-covalent bonds). For example, the antibodies of the present invention can be recombinantly fused or conjugated to molecules useful as labels in detection assays and to effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. For example, PCT Publication WO92 / 0849
5; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,9.
95; and EP 396,387.

The antibodies of the present invention may be modified (ie, covalently attached).
Attachment) includes derivatives (by covalent attachment of any type of molecule to the antibody) that do not prevent the antibody from producing an anti-idiotypic response. For example, without limitation, antibody derivatives include, for example, glycosylation, acetylation, pegylation, phosphorylation, and the like.
n), amidation, known protecting / blocking group
Antibodies that have been modified by derivatization, proteolytic cleavage, ligation to cellular ligands or other proteins, and the like. Numerous 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. Further, the derivative may contain one or more non-classical amino acids.

[0314] The antibodies of the present invention can be produced by any suitable method known in the art.
Polyclonal antibodies to an antigen of interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to a variety of host animals, including, but not limited to, rabbits, mice, rats, and the like, 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 BCG (bacilli Calmette-Guerin) and corynebacterium parvum
Including but not limited to potentially useful human adjuvants. Such adjuvants are also well-known in the art.

[0315] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using the following hybridoma techniques known in the art and include, for example, Harr
low et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press
Hammerling et al., Monoclonal An.
tibodies and T-Cell Hybridomas 563-6
81 (Elsevier, NY 1981), the above references being 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 derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method derived from the method by which it was generated.

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, a mouse can be immunized with a polypeptide of the invention or a cell expressing such a peptide. Once immune application is detected (eg, antigen-specific antibodies are detected in mouse serum)
The mouse spleen is collected and the spleen cells are isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells (eg, cells from cell line SP20 available from the ATCC). Hybridomas are selected and cloned by limiting dilution. Next, the hybridoma clones are assayed for cells secreting antibodies capable of binding the polypeptide of the invention by methods known in the art. Ascites fluid containing generally high levels of antibodies
) By immunizing mice with a positive hybridoma clone,
Can be produced.

Accordingly, the present invention provides a method of 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 method comprises the steps of: The hybridoma is obtained by fusing myeloma cells with splenocytes isolated from a mouse immunized with the antigen of the present invention, and then hybridoma clones secreting antibodies capable of binding to the polypeptide of the present invention.
It is produced by screening hybridomas resulting from the fusion.

[0318] Antibody fragments that recognize a particular epitope can be generated by known techniques. For example, Fab and F (ab ') 2 fragments of the present invention comprise (Fa
It can be produced by proteolytic cleavage of immunoglobulin molecules using an enzyme such as papain (to produce the b-fragment) or pepsin (to produce the F (ab ') 2 fragment). 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 an antigen binding domain expressed from a reper trimer 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 beads). The phage used in these methods is typically a phage having the antibody domain of a Fab, Fv or disulfide stabilized Fv recombinantly fused to either the phage gene III protein or the 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. Immuno
l. Methods 182: 41-50 (1995); Ames et al. Im
munol. Methods 184: 177-186 (1995); Kett.
leborough et al., Eur. J. Immunol. 24: 952-958 (
1994); Persic et al., Gene 187 9-18 (1997); Bu.
rton et al., Advances in Immunology 57: 191-.
280 (1994); PCT Application No. PCT / GB91 / 01134; PCT Publication WO 90/02809; WO 91/10737; WO 92/01047.
WO 92/18619; WO 93/11236; WO 95/15982
WO 95/20401; and US Pat. No. 5,698,426;
No. 5,223,409; No. 5,403,484; No. 5,580,717;
No. 5,427,908; No. 5,750,753; No. 5,821,04
No. 7, No. 5,571,698; No. 5,427,908; No. 5,516
No. 5,637, No. 5,780,225; No. 5,658,727;
Nos. 733,743 and 5,969,108, each of which is hereby incorporated by reference in its entirety.

As described in the above references, after phage selection, the regions encoding phage-derived antibodies may be used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment. Can be isolated and used, 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, an antibody having a variable region derived from a mouse 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; U.S. Pat.
Nos. 807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety.
. A humanized antibody is an antibody molecule 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 framework regions from a human immunoglobulin molecule. . Often, framework residues within the human framework regions are replaced with corresponding residues from a CDR donor antibody to alter (preferably improve) antigen binding. These framework substitutions are identified by methods well known in the art, and include, for example, the use of Cs to identify framework residues important for antigen binding.
By modeling the interaction of DR and framework residues, and by sequence comparison to identify abnormal framework residues at specific positions. (For example,
Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Na.
332: 323 (1988), which are incorporated herein by reference in their entirety. ) Antibodies can be humanized using various techniques known in the art, including: CDR-grafting (EP 239).
PCT Publication WO 91/09967; US Patent No. 5,225,53.
9, No. 5,530,101 and 5,585,089), veneering or resurfacin.
g) (European Patents 592,106; 519,596; Padlan, M.
molecular Immunology 28 (4/5): 489-498 (
1991); Studnicka et al., Protein Engineerin.
g 7 (6): 805-814 (1994); Roguska et al., PNAS 9
1: 969-973 (1994)), and chain shuffling (chai).
n shuffling) (U.S. Patent No. 5,565,332).

[0324] Fully 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 and 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.

[0323] Human antibodies can also be produced using transgenic mice, which are unable to express functional endogenous immunoglobulin, 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 randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region (diversity)
region) can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of a human immunoglobulin locus by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen (eg, all or a portion of a polypeptide of the invention). Monoclonal antibodies directed against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgene retained in the transgenic mouse is B
Rearranges during cell differentiation and subsequently undergoes class switching and somatic mutation. Thus, the use of such techniques allows for 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, as well as protocols for producing such antibodies, see, eg, PCT Publication WO 98/24893; WO 92/01047; WO 96/340.
WO 96/33735; EP 0 598 877; US Pat.
Nos. 413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806.
No. 5,814,318; No. 5,885,793; No. 5,916,
No. 771; and 5,939,598, which are incorporated herein by reference in their entirety. Further, Abgenix, Inc. (F
Companies such as Reemont, CA) and Genpharm, San Jose, CA may engage in providing human antibodies to the selected antigen using techniques similar to those described above.

Human antibodies that fully recognize the selected epitope were identified as “guided (gui
ded) selection ". In this approach,
Selected non-human monoclonal antibodies (eg, mouse antibodies) are used to guide the selection of human antibodies that fully recognize the same epitope. (Jespers
Et al., Bio / technology 12: 899-903 (1988)).

In addition, antibodies to the polypeptides of the invention may in turn be utilized to raise anti-idiotype antibodies that "mimic" the polypeptides of the invention using techniques well known to those of skill in the art. (Eg, Greenspan and Bona, FASEB
J. 7 (5): 437-444; (1989) and Nissinoff, J.
. Immunol. 147 (8): 2429-2438 (1991). E.g., bind and multimerize polypeptides
) and / or antibodies that competitively inhibit the binding of the polypeptide of the invention to its ligand are used to produce anti-idiotypes that "mimic" the multimerization and / or binding domains of the polypeptide. And thus binds 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 regimes to neutralize polypeptide ligand. For example, such anti-idiotype antibodies can be used to bind 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 of the present invention and a fragment thereof. The present invention also provides antibodies (preferably as specifically defined above) under stringent or lower stringency hybridization conditions (preferably, which specifically bind to a polypeptide of the present invention, preferably , An antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO: Y).

[0327] 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 the antibody can be constructed from chemically synthesized oligonucleotides (eg, Kutmeie).
et al., BioTechniques 17: 242 (1994), which, in brief, involves the following steps: Synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody. Annealing and ligation of these oligonucleotides, followed by amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody can be produced from nucleic acid from a suitable source. If no clone is available containing the nucleic acid encoding the particular antibody, but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from 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, to identify a cDNA clone from a cDNA library encoding the antibody, using PCR amplification using synthetic primers capable of hybridizing to the 3 'and 5' ends of the sequence. Or use oligonucleotide probes specific for a particular gene sequence. It may be obtained by cloning. P
The amplified nucleic acid generated by the CR can then be cloned into a replicable cloning vector using any method known in the art.

Once the nucleotide sequence and the corresponding amino acid sequence of the antibody have been determined,
The nucleotide sequence of an antibody can be determined by methods known in the art for manipulating nucleotide sequences (eg, recombinant DNA technology, 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., Eds. 1998, Current Protocols.
in Molecular Biology, John Wiley & Sons
, NY. These are both incorporated herein by reference in their entirety. )) Can be used to generate antibodies with different amino acid sequences to make, for example, amino acid substitutions, deletions, and / or insertions.

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

Furthermore, by splicing genes from mouse antibody molecules of appropriate antigen specificity with genes from human antibody molecules of appropriate biological activity,
Techniques 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 noted above, a chimeric antibody is a molecule in which different portions are derived from different animal species, such molecules having variable regions derived from the constant regions of mouse mAbs and human immunoglobulins (eg, humanized antibodies). .

Alternatively, techniques described for the production of single-chain antibodies (US Pat. No. 4,946,946)
No. 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)) may 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. FIG. Techniques for the assembly of functional Fv fragments in E. coli can also be used (Sk
erra et al., Science 242: 1038-1041 (1988)).

Methods for Producing Antibodies The antibodies of the invention can be produced by any method known in the art for synthesizing antibodies, particularly by chemical synthesis, or preferably, by recombinant expression techniques. obtain.

An antibody of the invention, or a fragment, derivative or analog thereof (eg,
Recombinant expression of a heavy or light chain of an antibody of the invention or a single chain antibody of the invention) requires the construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding the antibody molecule or antibody heavy or light chain, or portions thereof, (preferably containing the heavy or light chain variable domains) of the invention is obtained, the production of the antibody molecule is Can be produced by recombinant DNA technology 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 antibody-encoding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, the present invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the present invention, or a heavy or light chain thereof, or a variable domain of a heavy or light chain, operably linked to a promoter. I will provide a.
Such vectors contain the constant region of the antibody molecule (eg, PCT Publication WO86 /
05807; PCT Publication WO 89/01036; and US Pat. No. 5,122.
, 464), and the variable domains of the antibody can be cloned into such a vector for expression of the entire heavy or light chain.

The expression vector is transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce an antibody of the present invention. Accordingly, the invention includes host cells comprising a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In a preferred embodiment for expression of a double-chain antibody, the vector encoding both the heavy and light chains is 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 can be utilized to express the antibody molecules of the present invention. Such host expression systems represent vehicles in which the coding sequence of interest can be produced and subsequently purified, but also can be used in situ when transformed or transfected with sequences encoding the appropriate nucleotides. 1 represents a cell capable of expressing the 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 antibody-encoding sequences (eg, E. coli, A microorganism such as B. subtilis; a yeast (eg, Sacc) transformed with a recombinant yeast expression vector containing a sequence encoding the antibody.
haromyces, Pichia); an insect cell line infected with a recombinant virus expression vector (eg, baculovirus) containing a sequence encoding the antibody; a recombinant virus expression vector (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) A plant cell line infected with E. coli 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, a mammalian cell line (eg, COS, CHO, BHK) carrying a recombinant expression construct containing 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 E. coli, and more preferably, eukaryotic cells, particularly for the expression of whole recombinant antibody molecules, are used for expression of recombinant antibody molecules. For example, mammalian cells, such as Chinese hamster ovary cells (CHO), combined with vectors, such as the major immediate-early gene promoter element from human cytomegalovirus, are effective expression systems for antibodies. (Foecking et al., Gene 45: 101 (1986); Cock)
ett et al., Bio / Technology 8: 2 (1990)).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the expression of the antibody molecule. For example, if large quantities of such a protein are to be produced, a vector that directs high-level expression of a fusion protein product that is easily purified may be desirable for the production of a pharmaceutical composition of the antibody molecule. Such vectors include, but are not limited to, the following: the sequence encoding the antibody is in frame with the region encoding lacZ in the vector.
e) can be separately ligated, 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)). The pGEX vector can also be used as a fusion protein with glutathione S-transferase (GST) to express foreign polypeptides. Generally, 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 a thrombin or factor Xa protease cleavage site so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing heterologous genes. This virus grows in Spodoptera frugiperda cells. Sequences encoding the antibody can be cloned individually into non-essential regions of the virus, such as the polyhedrin gene, and placed under the control of an AcNPV promoter, such as the polyhedrin promoter.

In mammalian host cells, a number of viral-based expression systems may 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, such as the late promoter and a three-part leader sequence. Then
This chimeric gene can be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertions in non-essential regions of the viral genome (eg, the E1 or E3 regions) result in a recombinant virus that is viable and capable of expressing the antibody molecule in the infected host (eg, Logan & Shenk)
Proc. Natl. Acad. Sci. USA 81: 355-359 (1
984)). Specific initiation signals may also be required for efficient translation of the inserted antibody-encoding sequence. These signals are ATG
Includes start codon and adjacent sequences. In addition, this initiation codon may be used to ensure that the translation of the entire insert is in reading frame of the desired coding sequence (eg,
reading frame). 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 may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
Such modifications (eg, glycosylation) and processing (eg,
For example, cleavage) can 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 heterologous protein expressed. To this end, eukaryotic host cells that have the cellular machinery for the correct processing of first transcription, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, WI38, and especially, 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, for example, CRL7030 and Hs578Bst.

[0341] Long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the antibody molecule can be engineered. Rather than using an expression vector that contains the viral origin of replication, the host cell may have appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.).
, And DNA controlled by a selectable marker. Following the introduction of the heterologous DNA, the engineered cells can be allowed to grow for 1-2 days in an enriched media, and are then switched to a selective media. The selectable marker on the recombinant plasmid confers resistance to the selection and allows the cell to stably integrate the plasmid into its chromosome and grow to form a cell foci, which in turn can be cloned. , Allowing it to be expanded into cell lines. This method can be advantageously used to engineer cell lines that express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluating compounds that interact directly or indirectly with the antibody molecule.

A number of selection systems can be used, such as 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
Owy et al., Cell 22: 817 (1980)), which can be used in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as a basis for selection of the following genes: dhfr, which confers 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-
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, Sc.
issue 260: 926-932 (1993); and Morgan an.
d Anderson, Ann. Rev .. Biochem. 62: 191-21
7 (1993); May 1993, TIB TECH 11 (5): 155-2.
15); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30: 147 (1984)). Recombinant DNA
Methods well known in the art can be routinely applied to select a desired recombinant clone, and such methods are described below:
ubel et al. (eds.), Current Protocols in Molecu.
lar Biology, John Wiley & Sons, NY (1993)
Kriegler, Gene Transfer and Expressi;
on, A Laboratory Manual, Stockton Pres
s, NY (1990); and Chapters 12 and 13, Dracopoli et al. (
Ed.), Current Protocols in Human Geneti
cs, John Wiley & Sons, NY (1994);
-Garapin et al. Mol. Biol. 150: 1 (1981), which are hereby incorporated by reference in their entirety.

The expression level of an antibody molecule can be increased by vector amplification (for review, see
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 culture of the host cell will increase the number of copies of the marker gene. Since the augmented region is associated with the antibody gene, production of the antibody 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 polypeptide derived from the heavy chain and a second vector encoding a polypeptide derived from the light chain. . The two vectors may contain the same selectable marker that allows for equal expression of the 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 a situation,
The light chain should be placed before the heavy chain to avoid excessive toxic free heavy chains (Proudfoot, Nature 322: 52 (1986); Kohle
r, Proc. Natl. Acad. Sci. USA 77: 2197 (198
0)). The coding sequences for the heavy and light chains can include cDNA or genomic DNA.

Once an antibody molecule of the present invention has been produced by an animal, chemically synthesized, or recombinantly expressed, any of the methods known in the art for the purification of immunoglobulin molecules are available. Methods, 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, solubility differences, or any other standard technique for protein purification.
In addition, the antibodies or fragments thereof of the present 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), which are recombinantly fused or chemically linked (including both covalent and non-covalent bonds) to produce a fusion protein. The fusion need not be direct, but can occur via a linker sequence. The antibody comprises 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, a polypeptide of the invention can be fused or conjugated to an antibody specific for a particular cell surface receptor to bind the polypeptide of the invention to a particular cell type. To target
Antibodies can be used. Antibodies fused or conjugated to the polypeptides of the present invention can also be used in in vitro immunoassays and purification methods using methods known in the art. See, for example, Harbor et al., Supra, and PCT Publication WO 93.
EP 439,095; Naramura et al., Immunol. L
ett. 39: 91-99 (1994); U.S. Patent No. 5,474,981; G
illies et al., PNAS 89: 1428-1432 (1992); Fell.
J. et al. Immunol. 146: 2446-2452 (1991). These are incorporated by reference in their entirety.

The present invention further includes compositions comprising the polypeptides of the present invention fused or linked to domains other than the variable regions of the antibody. For example, a polypeptide of the invention can be fused or conjugated to the Fc region of an antibody, or a portion thereof. The portion of this antibody fused to the polypeptide of the invention comprises the constant region, hinge region, CH1 domain, C
It may 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 portions of the above-described antibodies, forming a multimer. For example, an Fc portion fused to a polypeptide of the invention can form a dimer through disulfide bonds between the Fc portions. Higher multimeric forms convert polypeptides to IgA
And by fusion to portions of IgM. Methods for fusing or binding the polypeptides of the present invention to an antibody moiety are known in the art. For example, U.S. Patent Nos. 5,336,603; 5,622,929;
No. 5,359,046; No. 5,349,053; No. 5,447,85
No. 5,112,946; EP 307,434; EP 367,16
6; PCT Publication WO96 / 04388; WO91 / 06570; Ashkena
zi et al., Proc. Natl. Acad. Sci. USA 88: 10535-
10439 (1991); Zheng et al. Immunol. 154: 559
0-5600 (1995); and Vil et al., Proc. Natl. Acad.
Sci. USA 89: 113337-11341 (1992), which is incorporated by reference in its entirety.

As discussed above, a polypeptide corresponding to a polypeptide, polypeptide fragment, or variant of SEQ ID NO: Y may be used to increase the in vivo half-life of the polypeptide, or The antibodies can be fused or conjugated to the above antibody moieties for use in immunoassays using methods known in the art. In addition, a polypeptide corresponding to SEQ ID NO: Y may be fused or conjugated to the above antibody portion to facilitate purification. One reported example describes a chimeric protein consisting of the first two domains of a human CD4 polypeptide and various domains of the constant regions of the heavy or light chains of a mammalian immunoglobulin (EP 394). ,
827; Traunecker et al., Nature 331: 84-86 (198
8)). The polypeptides of the present invention, which are fused or conjugated to an antibody having a disulfide-linked dimeric structure (due to IgG), also bind to other molecules than the monomeric secreted protein or protein fragment alone, and It may be more efficient to neutralize (Funtoulakis et al., J. Biochem. 27).
0: 3958-3964 (1995)). In many cases, the Fc portion of the fusion protein is beneficial in therapy and diagnosis, and thus may 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, if the 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 an Fc portion 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. Biol. Che
m. 270: 9449-9471 (1995)).

Further, the antibodies or fragments thereof of the present invention can be fused to a marker sequence, such as a peptide to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexa-histidine peptide, such as a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chats).
tags provided in worth, CA, 91311), 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 for convenient purification of the fusion protein. Another peptide tag useful for purification is the “HA” tag corresponding to an epitope from the influenza hemagglutinin protein (Wilson et al., Ce.
1137: 767 (1984)), and the "flag" tag.

The present invention further includes an antibody or fragment thereof conjugated to a diagnostic or therapeutic agent. 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 coupling 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 , And the like. The detectable substance is an antibody (or fragment thereof)
Can be linked or attached, either directly or indirectly, via an agent (eg, a linker known in the art) using techniques 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 substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent substances include luminol Examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive materials include 125I, 131I, 111In or 99Tc.

Further, the antibody or fragment thereof may be a therapeutic moiety (eg, a cytotoxin (eg, a cytostatic or cytocidal agent)), a therapeutic agent, or a radiometal ion (eg, an α-emitter- (eg, 213Bi)). Etc.). Cytotoxic or cytotoxic agents include any agents that are harmful to cells. Examples include paclitaxol, cytochalasin B, gramicidin D,
Ethidium bromide, emethine, mitomycin, etoposide, teniposide (ten
oposide), vincristine, vinblastine, colchicine (colch)
icin), doxorubicin, daunorubicin, dihydroxy anthracin 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
fluorouracil decarbazine), an alkylating agent (for example,
Mechlorethamine, thioepa chlorambucil, melphalan,
Carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin, anthracycline (eg, daunorubicin) (Formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and antimitotic agents (eg, vincristine and vinblastine) ), But
Not limited to them.

The conjugates of the present invention can be used to modify a given biological response, and the therapeutic or drug moiety is not to be construed as limited to classical chemotherapeutic 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, α-interferon, β-interferon, nerve growth factor,
Platelet-derived growth factor, tissue plasminogen activator, apoptotic drug (eg, TNF-α, TNF-β, AIMI (International Publication WO 97/33899)
AIM II (see WO 97/34911), Fas ligand (Takahashi et al., Int. Immunol. 6).
: 1567-1574 (1994)), VEGI (International Publication No. WO 99/231).
No. 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).

Antibodies can also be attached to a solid support, which is particularly useful for immunoassays 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 and include, for example, Arnon
Et al., "Monoclonal Antibodies For Immunot."
arranging Of Drugs In Cancer Therapy "
Monoclonal Antibodies And Cancer The The
rapy, Reisfeld et al. (eds.), pages 243-56 (Alan R. Lis.
s, Inc. 1985); Hellstrom et al., "Antibodies F.
or Drug Delivery "Controlled Drug Del
Ivery (second edition), Robinson et al. (eds.), pp. 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
pp. 475-506 (198).
5); “Analysis, Results, And Future Pros
sensitive of therapeutic use of ra
diolable Antibody In Cancer Therap
y "Monoclonal Antibodies For Cancer D
selection And Therapy, Baldwin et al. (eds.), 303
-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Prop."
arts of Antibody-Toxin Conjugates "
, Immunol. Rev .. 62: 119-58 (1982).

Alternatively, the antibody is conjugated to a secondary antibody, and antibody heterologous binding as described by Segal in US Pat. No. 4,676,980, which is incorporated by reference herein in its entirety. Heterojugates may be formed.

Antibodies 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 therapeutics.

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

[0358] These techniques are useful for hematological malignancies (ie, minimal residual disease (MRD) in patients with acute leukemia).
And screening of specific populations of cells as can be found with "non-self" cells in transplantation to prevent graft versus host disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells that can undergo proliferation and / or differentiation as can be found in human cord blood.

Assays for Antibody Binding Antibodies of the invention can be assayed for immunospecific binding by any method known in the art. Immunoassays that can be used include Western blots, radioimmunoassays,
ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, sedimentation reaction, gel diffusion sedimentation reaction, immunodiffusion assay, agglutination assay, complement binding assay, immunoradiometric assay, fluorescent immunoassay, protein A Examples include, but are not limited to, competitive and non-competitive assay systems that use techniques such as immunoassays. Such assays are conventional and well known in the art (eg, Ausubel et al., Eds., 1994, Current Protocol, which is incorporated by reference herein in its entirety).
ls in Molecular Biology, Volume 1, John Wil
eye & Sons, Inc. , New York). Exemplary immunoassays are briefly described below (although these are not intended to be limiting).

The immunoprecipitation protocol generally involves RIPA buffer (1% NP-40 or Triton X-100, 1% NP-40 or Triton X-100, supplemented with a protein phosphatase inhibitor and / or a protease inhibitor (eg, EDTA, PMSF, aprotinin, sodium vanadate)). % Sodium deoxycholate, 0.1%
SDS, 0.15 M NaCl, 0.01 M sodium phosphate (pH 7.2)
) Lysing a population of cells in a lysis buffer such as 1% Trasylol, adding the antibody of interest to the cell lysate, and incubating at 4 ° C. for a certain period of time (eg, 1-4 hours). Adding protein A sepharose beads and / or protein G sepharose beads to the cell lysate, incubating at 4 ° C. for about 1 hour or more, washing the beads in lysis buffer, and SDS / sample buffer Resuspending the beads in the solution. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assayed, for example, by Western blot analysis. One skilled in the art can appreciate for parameters that can be modified to increase the binding of the antibody to the antigen and reduce the background, such as the step of pre-clearing the cell lysate using Sepharose beads. . For further discussion on immunoprecipitation protocols, see, for example, Ausub
el et al., 1994, Current Protocols in Molec.
ullar Biology, Volume 1, John Wiley & Sons, Inc.
. , New York, 10.16.1.

Western blot analysis generally involves the steps of preparing a protein sample, polyacrylamide gels (eg, 8% -20% SDS-depending on the molecular weight of the antigen).
Electrophoresis of the protein sample in PAGE), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, a blocking solution (eg, PBS with 3% BSA or skim milk powder) Blocking the membrane in a washing buffer (eg, PB
Washing the membrane in S-Tween 20), blocking the membrane with a primary antibody (antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer A secondary antibody (recognizing a primary antibody, eg, an anti-human antibody) coupled to an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (eg, 32P or 125I) diluted in blocking buffer To block the membrane, wash the membrane in a wash buffer, and detect the presence of the antigen. One skilled in the art will be aware of parameters that can be modified to increase the signal detected and reduce background noise. For further discussion on Western blot protocols, see, for example, Ausu
Bel et al., 1994, Current Protocols in Mole.
Cultural Biology, Volume 1, John Wiley & Sons, In
c. , New York, 10.8.1.

The ELISA involves preparing the antigen, coating the wells of a 96-well microtiter plate with the antigen, binding to a detectable compound such as an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase). Adding the antibodies to the wells and incubating for a period of time, and detecting the presence of the antigen. In an ELISA, the antibody of interest need not be conjugated to a detectable compound; instead, a second antibody (recognizing the antibody of interest) conjugated to the detectable compound can be added to the wells. . Further, instead of coating the well with the antigen, the antibody can be coated on the well. In this case, a second antibody bound to the detectable compound can be added subsequent to the addition of the antigen of interest to the coated wells. One skilled in the art will be aware of parameters that can be modified to increase the signal detected, and other variations of ELISAs known in the art. EL
For further discussion on ISA, see, for example, Ausubel et al., Eds., 199.
4, Current Protocols in Molecular Bio
logic, Volume 1, John Wiley & Sons, Inc. , New Yo
See rk, 11.2.1.

The binding affinity of the antibody for 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 involves incubating a labeled antigen (eg, 3H or 125I) with an antibody of interest in the presence of increasing amounts of unlabeled antigen, and labeling. Includes detection of antibody bound to 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 incubated with the antibody of interest conjugated to a labeled compound (eg, 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses The present invention further relates to antibody-based therapies, wherein the therapies are used to treat one or more of the disclosed diseases, disorders or conditions in animals, preferably mammals, and most Preferably, the method includes the step of administering the antibody of the present invention to a human patient. Therapeutic compounds of the invention include 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 may comprise a disease, disorder or condition associated with abnormal expression and / or activity of a polypeptide of the invention (including any one or more diseases, disorders or conditions described herein). But not limited thereto) can be used to treat, inhibit or prevent. Abnormal expression of the polypeptide of the invention and / or
Or treating and / or preventing a disease, disorder or condition associated with an activity includes, but is not limited to, alleviating the symptoms associated with the disease, disorder or condition. Antibodies of the invention can be provided in pharmaceutically acceptable compositions, as known in the art or as described herein.

A summary of how the antibodies of the present invention may be used therapeutically is mediated locally or systemically in the body, or (eg, by complement (CDC) or by effector cells (ADCC). Binding the polynucleotide or polypeptide of the present invention, due to the direct cytotoxicity of the antibody. Some of these approaches are described in more detail below. Given the teachings provided herein, one of skill in the art would recognize, without undue experimentation, how to use the antibodies of the present invention for diagnostic, monitoring, or therapeutic purposes. Understand.

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

The antibodies of the invention can be administered alone or in combination with other types of treatment, such as radiation therapy, chemotherapy, hormonal therapy, immunotherapy and antitumor agents.
Generally, administration of a species-derived or species-reactive product that is the same species as the patient (in the case of antibodies) is preferred. Thus, in a preferred embodiment, a human antibody,
Fragment derivatives, analogs, or nucleic acids are administered to a human patient for treatment or prevention.

[0368] High affinity and / or high affinity polypeptides or polynucleotides of the present invention for both immunoassays for the polynucleotides or polypeptides of the present invention (including fragments thereof), and treatment of disorders related thereto. Or potent, in vivo inhibitory and / or neutralizing antibodies, fragments thereof,
Alternatively, it is preferable to use those regions. Such an antibody, fragment or region is preferably a polynucleotide or polypeptide (
(Including its fragments). Preferred binding affinities include 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 × 10 -13 M
Binding affinities having a dissociation constant or Kd of less than 10, 10 ^-13 M, 5 x 10 ^-14 M, 10 ^-14 M, 5 x 10 ^-15 M, and 10 ^-15 M.

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

[0370] Any of the methods for gene therapy available in the art can be used according to the present invention. An exemplary method is described below.

For a general review of methods for gene therapy, see Goldspiel et al., Cli.
natural 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.
TECH 11 (5): 155-215 (1993). Methods generally known in the recombinant DNA art that can be used are Ausubel
Et al. (Ed.), Current Protocols in Molecular
Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expressio.
n, A Laboratory Manual, Stockton Press
, NY (1990).

In a preferred aspect, the compound comprises a nucleic acid sequence encoding an antibody, wherein the nucleic acid sequence is an expression vector that expresses the antibody, or a fragment or chimeric protein thereof, or a heavy or light chain thereof, in a suitable host. Part of. In particular, such nucleic acid sequences have a promoter operably linked to the antibody coding region, wherein the promoter is inducible or constitutive, and optionally tissue-specific. In another particular embodiment, a nucleic acid molecule is used in which the sequence encoding the antibody and any other desired sequences are flanked by regions that promote homologous recombination at the desired site in the genome, thus rendering the antibody Provides intrachromosomal expression of the encoding nucleic acid (Koller and Smithies, Proc. N
atl. 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 a nucleic acid to a patient can be direct (where the patient is directly exposed to the nucleic acid or nucleic acid-bearing vector) or indirect (where the cells are first transduced with the nucleic acid in vitro). Converted and then implanted into the patient). These two approaches are known, respectively, as in vivo gene therapy or as ex vivo gene therapy.

In certain embodiments, a nucleic acid sequence is administered directly in vivo, where the nucleic acid sequence is expressed to produce an encoded product. This involves a number of methods known in the art, such as constructing them as part of a suitable nucleic acid expression vector, and then constructing it (eg, a defective or attenuated retrovirus or other viral vector (US Pat. No. 4,980,286), by intracellular administration, or by direct injection of naked DNA, or by microparticle bombardment (eg, a gene gun; Biol.
istic, Dupont), or by coating with lipids or cell surface receptors, or by transfecting the drug, or by encapsulation in liposomes, microparticles, or microcapsules, or by nucleating them. By conjugation with a peptide known to enter into the cell, and by conjugation with a ligand that undergoes receptor-mediated endocytosis (eg, Wu and Wu, J. 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 fusogen that disrupts endosomes.
ic) a viral peptide, which enables this nucleic acid to avoid lysosomal degradation. In yet another embodiment, nucleic acids can be targeted in vivo for cell-specific uptake and expression by targeting specific receptors (eg, PCT Publication Nos. WO92 / 06180; WO92 / 226).
No. 35; WO92 / 20316; WO93 / 14188, WO
93/20221). Alternatively, the nucleic acid can be introduced inside the cell and integrated into host cell DNA for expression by homologous recombination (
See Koller and Smithies, Proc. Natl. Acad. Sci
. ScL USA 86: 8932-8935 (1989); Zijlstra et al., Na.
cure. 342: 435-438 (1989)).

In certain embodiments, a viral vector that includes a nucleic acid sequence encoding an antibody of the invention is used. For example, retroviral vectors can be used (Mi
ller et al., Meth. Enzymol. 217: 581-599 (1993)
checking). These retroviral vectors contain the necessary components for correct packaging of the viral genome and correct integration into host cell DNA. Nucleic acid sequences encoding antibodies used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. Further details on retroviral vectors can be found in Boesen et al., Bi.
other, see the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make the stem cells 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);
almons and Gunzberg, Human Gene Therapy
4: 129-141 (1993); and Grossman and Wils.
on, Curr. Opin. in Genetics and Level. 3
: 110-114 (1993).

[0367] 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. Adenovirus has the advantage that it can infect non-dividing cells. Kozarsky and Wilson, Curr
ent Opinion in Genetics and Development
ent 3: 499-503 (1993) provides an overview of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5: 3.
-10 (1994) showed the use of adenovirus vectors to transfer genes to the airway epithelium of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy include Rosenfeld et al., Science 252: 431-434 (19).
91); Rosenfeld et al., Cell 68: 143-155 (1992).
Mastranelli et al., J .; Clin. Invest. 91: 225-2
34 (1993); PCT Publication No. WO 94/12649; and Wang et al.,
Gene Therapy 2: 775-783 (1995).
In a preferred embodiment, an adenovirus vector is used.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:
289-300 (1993); U.S. Patent No. 5,436,146).

Another approach to gene therapy involves transferring the 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 under selection to isolate cells that take up and express the transferred gene.
The cells are then delivered to the patient.

In this embodiment, the nucleic acid is introduced into the cells prior to in vivo administration of the resulting recombinant cells. Such introduction can be performed by any method known in the art, including, but not limited to: transfection, electroporation, microinjection, viruses including nucleic acid sequences. Vector or bacteriophage vector infection, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and the like. Numerous techniques are known in the art for introducing foreign genes into cells (eg, Loeffler and Behr, Met).
h. Enzymol. 217: 599-618 (1993); Cohen et al., M.
eth. Enzymol. 217: 618-644 (1993); Cline,
Pharmac. Ther. 29: 69-92m (1985)),
And if the necessary developmental and physiological functions of the recipient cells are not disrupted, they can be used in accordance with the present invention. The technique should provide for a stable transfer of the nucleic acid into the cell, so that the nucleic acid is expressible by the cell, and preferably is hereditary and expressible by the progeny of the cell.

[0380] 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 contemplated for use depends on the desired effect, patient condition, etc., and can be determined by one skilled in the art.

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

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

In embodiments where recombinant cells are used in gene therapy, the nucleic acid sequence encoding the antibody is introduced into the cell such that the nucleic acid sequence is expressible by the cell or progeny thereof, and then the recombinant cell is Administered in vivo for therapeutic effect. In a specific embodiment, stem cells or progenitor cells are used. Any stem and / or progenitor cells that can be isolated in vitro and maintained in vitro can potentially be used according to this embodiment of the invention (
For example, PCT Publication No. WO 94/08598: Temple and Ande
rson, Cell 71: 973-985 (1992); Rheinwald
, Meth. Cell Bio. 21A: 229 (1980); and Pit
Telkow and Scott, Mayo Clinic Proc. 61: 7
71 (1986)).

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

Demonstration of therapeutic or prophylactic activity The compounds or pharmaceutical compositions of the invention are preferably tested in vitro prior to use in humans and then in vivo for the desired therapeutic or prophylactic activity. Is done. 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 a cell line or patient tissue sample. The effect of a compound or composition on a 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 grows in culture, The compound is then exposed or otherwise administered, and the effect of such compound on the tissue sample is observed.

Therapeutic / Prophylactic Administration and Composition The present invention provides for treatment, inhibition and prevention by administering to a subject an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. To provide a way. In a preferred aspect, the compound is substantially purified (eg, substantially free of substances that limit its effect or produce undesired side effects). The subject is preferably
Animals including but 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 an immunoglobulin are described above; further suitable formulations and routes of administration are selected from those described herein below. Can be done.

Various delivery systems are known and can be used to administer the compounds of the invention (eg, liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing the compounds). , Receptor-mediated endocytosis (
See, for example, Wu and Wu, J. et al. Biol. Chem. 262: 4429-443
2 (1987)), construction of nucleic acids as part of retroviruses or other vectors). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition may be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucosal linings such as oral, rectal and intestinal mucosa, and other It can be administered together with a biologically active agent. Administration can be systemic or local. In addition, the pharmaceutical compounds or compositions of the present invention can be administered by any suitable route, including intraventricular and intrathecal injections; intraventricular injection can be, for example, an intraventricularly attached reservoir such as an Ommaya reservoir. It may be desirable to introduce it into the central nervous system (which can be facilitated by a catheter). Pulmonary administration may also be used, for example, with the 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 a limitation, for example, surgery. By local injection, topical application (eg, in combination with a post-operative wound dressing), by injection, by catheter, by suppository, or by an implant (this implant may be a membrane such as a sialastic membrane or Including fibers, which are porous, non-porous, or glue-like materials). Preferably, when administering a protein of the invention, including an antibody, care must be taken to use materials to which the protein does not absorb.

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

In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may 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. Engl. J. Med. 321:
574 (1989)). In another embodiment, a polymeric material may be used (Medical Applications of Control).
led Release, Langer and Wise (eds.), CRC Pre
s. , Boca Raton, Florida (1974); Control.
ed Drug Bioavailability, Drug Product
Design and Performance, Smolen and Bal
1 (eds), Wiley, New York (1984); Ranger and P.
eppas, J .; 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. Neurosurg. 71: 105 (
1989)). In yet another embodiment, the controlled release system can be placed near the therapeutic target, ie, the brain, and thus requires only a portion of the systemic dose (eg, Goodson, Medical Applicat).
ions of Controlled Release, (supra), Volume 2,
Pp. 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 protein-encoding nucleic acid, the nucleic acid is such that it is part of a suitable nucleic acid expression vector and is intracellular. By administration (eg, by use of a retroviral vector (see US Pat. No. 4,980,286)), or by direct injection, or by microparticle bombardment (eg, a gene gun; Biolis).
tic, Dupont) or by coating with lipids or cell surface receptors or transfectants, or in conjunction with a homeobox-like peptide known to enter the nucleus (eg,
Joliot et al., Proc. Natl. Acad. Sci. USA 88:18
64-1868 (1991)), and the like, to enhance the expression of the encoded protein. Alternatively, the nucleic acid can be introduced intracellularly and integrated into host cell DNA by homologous recombination for expression.

[0394] The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" has been approved by the Federal regulatory authority or the U.S. Government for use in animals, and more particularly in humans, or Means listed in the recognized pharmacopeias.
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, plant, or synthetic origin (eg, peanut oil, soybean oil, mineral oil, sesame oil, and the like). 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, skim milk powder, glycerol, propylene , Glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, 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 saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in W. "Reming by Martin
ton's Pharmaceutical Sciences ". Such compositions provide a therapeutically effective amount of the compound, preferably in purified form,
Included with an appropriate amount of carrier provides a form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated according to conventional procedures, as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the components are either separated or mixed together in a single dosage form, for example, in an ampoule or sachet (s) representing a fixed amount of active agent.
Supplied as a lyophilized powder or water-free concentrate in a sealed container such as an acetette. If the composition is to be administered by infusion, the composition can be dispensed into infusion bottles containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, the components can be mixed prior to administration.
An ampoule of sterile water for injection or saline can be provided.

The compounds of the present invention may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids and the like, and sodium, potassium, ammonium, calcium, ferric hydroxide, Isopropylamine, triethylamine,
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 abnormal expression and / or activity of a polypeptide of the invention) is determined by standard clinical techniques. Can be done. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be used in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves obtained from in vitro or animal model test systems.

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

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

Diagnostics and Imaging Labeled antibodies that specifically bind to a polypeptide of interest, and derivatives and analogs thereof, are used for diagnostic purposes to detect abnormal expression of a polypeptide of the invention and its expression. Diseases and / or disorders associated with / or activity may be detected, diagnosed or monitored. The present invention provides for the detection of abnormal expression of a polypeptide of interest, comprising the steps of (a) using one or more antibodies specific for the polypeptide of interest in a cell or body fluid of an individual. Assaying for the expression of the polypeptide of
And (b) comparing the gene expression level to a standard gene expression level, thereby increasing or decreasing the assayed polypeptide gene expression level compared to the standard expression level. Show 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 cells or body fluids of an individual using one or more antibodies specific for the polypeptide of interest; and (b) Comparing the level of gene expression, whereby an increase or decrease in the assayed polypeptide gene expression level compared to its standard expression level is indicative of a particular disorder. For cancer, the presence of relatively high amounts of transcripts in biopsy tissue from an individual may indicate a predisposition for the development of the disease, or provide a means for detecting the disease before the actual clinical symptoms appear. Can provide. A more conclusive diagnosis of this type may allow health professionals to use preventive measures, or allow early aggressive treatment, thereby preventing the development or further progression of cancer.

The antibodies of the present invention can be used to assay protein levels in biological samples using classical immunohistological methods known to those of skill in the art (eg, Jalk).
anen et al. Cell. Biol. 101: 976-985 (1985);
Jalkanen et al. Cell. Biol. 105: 3087-3096 (
1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art, and are enzymatic labels (eg, glucose oxidase); radioisotopes (eg, iodine (125I,
21I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc)); luminescent labels (eg, luminol); and fluorescent labels (eg, fluorescein and rhodamine); Biotin.

One aspect of the present 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, the diagnosis comprises: a) administering to the subject (eg, parenterally, subcutaneously, or intraperitoneally) an effective amount of a labeled molecule that specifically binds the polypeptide of interest. B) to allow the labeled molecule to preferentially concentrate 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, such that the label exceeds the background level. Detection of an activating molecule indicates that the subject has a particular disease or disorder associated with abnormal expression of the polypeptide of interest. Background levels can be determined by a variety of methods, including comparing the amount of labeled molecule detected with standard values previously determined for a particular system.

It is understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety required to generate a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected will usually be about 5-
It is in the range of 99 mTc of 20 millicuries. The labeled antibody or antibody fragment is then preferentially accumulated at locations in the cell that contain the particular protein.
In vivo tumor imaging is described by S.D. W. Burchiel et al., "Immunophar.
mackinetics of Radiolabeled Antibod
is and Their Fragments "(Tumor Image
ng: The Radiochemical Detection of Ca
Chapter 13, S.Ncer. W. Burchiel and B.A. A. Rhodes, Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and the mode of administration, the labeled molecule will preferentially concentrate at the site of the subject and the unbound labeled molecule Is between 6 and 48 hours or between 6 and 24 hours or between 6 and 12 hours after which administration can be cleared to background levels.
In another embodiment, the time interval after administration is 5-20 days or 5-10 days.

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

[0407] The presence of a labeled molecule can be detected from a patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. One of skill in the art can determine the appropriate method for detecting a particular label. Methods and devices that can be used in the diagnostic methods of the invention include, but are not limited to, computerized tomography (CT), whole body scanning such as proton emission tomography (PET), magnetic resonance imaging (MRI). , And ultrasound diagnostics.

In certain embodiments, the molecule is labeled with a radioisotope and detected from the patient using a radioresponsive 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 device. In another embodiment, the molecule is labeled with a positron emitting metal and is detected from the patient using proton emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and detected from the patient using magnetic resonance imaging (MRI).

(Kit) The present invention provides a kit that can be used in the above method. In one embodiment, the kit comprises an antibody (preferably a purified antibody) of the invention in one or more containers. In certain embodiments, a kit of the invention comprises a substantially isolated polypeptide comprising an epitope that is specifically immunoreactive with the antibodies contained in the kit. Preferably, the kit of the present invention further comprises a control antibody that does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention provide for the binding of an antibody to a polypeptide of interest (eg, the antibody is a detectable substrate (eg, the antibody is a fluorescent compound, an enzyme substrate, a radioactive compound or (A second antibody that can be conjugated with a luminescent compound) or that recognizes the first antibody can be conjugated to a detectable substrate).

In another particular embodiment of the invention, the kit is used for screening diagnostic sera containing antibodies specific for proliferative and / or cancerous polynucleotides and polypeptides. It is a kit. Such a kit can include a control antibody that does not react with the polypeptide of interest. Such a kit may comprise a substantially isolated polypeptide antigen comprising an epitope specifically immunoreactive with at least one anti-polypeptide antigen antibody. In addition, such kits can be used to detect binding of the antibody to an antigen (eg, the antibody can be conjugated to a fluorescent compound such as fluorescein or rhodamine, which can be detected by flow cytometry). Means. In certain embodiments, the kit may 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 above kit comprises a solid support to which the polypeptide antigen is attached. Such kits also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the reporter-labeled antibody.

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

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

[0414] The solid surface reagents in the assays described above include a
For example, prepared by known techniques for attachment to polymeric beads, dipsticks, 96-well plates or filtration materials). These methods of attachment generally include non-specific adsorption of proteins to a support or chemically active groups (eg, activated carboxyl, hydroxyl, or aldehyde groups) on a solid support. Covalent attachment of the protein (typically via a free amine group). Alternatively, streptavidin-coated plates can be used with biotinylated antigen.

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

[0416] Fusion Proteins Any of the polypeptides of the invention can be used to produce fusion proteins. For example, a polypeptide of the invention can be used as an antigenic tag when fused to a second protein. Antibodies raised against the polypeptides of the present invention can be used to indirectly detect a second protein by binding to the polypeptide. In addition, since the secreted protein targets cellular locations 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 polypeptide of the present invention include not only a heterologous signal sequence but also other heterologous functional regions. The fusion need not be direct, but can occur via a linker sequence.

In addition, fusion proteins can also be engineered to improve the characteristics of the polypeptide of the invention. For example, additional amino acids, particularly regions of charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and durability during purification or subsequent handling and storage from host cells. Also, peptide moieties can 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 (including fragments, and especially epitopes) of the present invention may comprise immunoglobulin (IgA, IgE, IgG, IgM) constant domains or portions thereof (CH1, CH2, CH3, and any thereof). Combination of (
), Including both the entire domain and portions thereof)) to produce chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example is human CD4
A chimeric protein consisting of the first two domains of a polypeptide and various domains of the constant regions of the heavy or light chains of a mammalian immunoglobulin has been described (EPA 394,827; Traunecker et al., Nature 33).
1: 84-86 (1988)). Fusion proteins with a disulfide-linked dimeric structure (due to IgG) may also be more efficient at binding and neutralizing other molecules than monomeric secreted proteins or protein fragments alone (Funtoulakis et al.). J. Biochem. 270: 395.
8-3964 (1995)).

[0420] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869).
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 diagnostics, and thus may 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, if the 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 portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5 (D. Bennett et al., J. Molecular Reco
g. 8: 52-58 (1995); Johanson et al.
Biol. Chem. 270: 9449-9471 (1995)).
.

In addition, a polypeptide of the invention can be fused to a marker sequence, such as 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 by Chatsworth, CA, 91311).
Many of these marker amino acid sequences are commercially available. See, for example, Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1
As described in 989), hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein (Wi
lson et al., Cell 37: 767 (1984)).

Thus, any of these above fusions can be engineered using a polynucleotide or polypeptide of the present invention.

Vectors, Host Cells, and Protein Production The present invention also relates to vectors containing the polynucleotides of the invention, host cells, and the production of polypeptides by recombinant techniques. For example, the vector can be a phage, plasmid, viral, or retroviral vector. Retroviral vectors can be replication-competent or replication-defective. In the latter case, viral growth is generally complete.
occurs only in the host cell.

[0424] The polynucleotide may be ligated to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using a suitable packaging cell line and then transduced into host cells.

The polynucleotide insert may be a suitable promoter (eg, phage λPL promoter, E. coli lac promoter, trp promoter, phoA promoter and tac promoter, SV40 early promoter and SV40 late promoter, and a retrovirus, to name a few. LTR promoter). Other suitable promoters are known to those skilled in the art. The expression construct further comprises sites for transcription initiation, transcription termination,
And a ribosome binding site for translation in the transcribed region. The coding portion of the transcript expressed by this construct is preferably a translation initiation codon first and a termination codon (U
AA, UGA or UAG).

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418, or neomycin resistance gene for eukaryotic cell culture, and E. coli. Includes a tetracycline, kanamycin or ampicillin resistance gene for culturing in E. coli and other bacteria. Representative examples of suitable hosts include bacterial cells (eg, E. c.
oli, Streptomyces and Salmonella typhim
urium cells); fungal cells (eg, yeast cells); insect cells (eg, Dros)
opilia S2 and Spodoptera Sf9 cells); animal cells (
(E.g., CHO cells, COS cells, 293 cells, and Bowes melanoma cells); and plant cells. Suitable 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
Ript vector, Phasescript vector, pNH8A, pNH16
a, pNH18A, pNH46A (Stratagene Cloning S
systems, Inc. And ptrc99a, pKK223
-3, pKK233-3, pDR540, pRIT5 (Pharmacia B
iotech, Inc. Available from Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (S
available from Tratagene); and pSVK3, pBPV, pMSG
And pSVL (available from Pharmacia). Other suitable vectors will be readily apparent to one skilled in the art.

The introduction of the construct into the host cell can be performed by calcium phosphate transfection, DE
It can result from AE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such a method is described in Davis et al., Basic Metho.
It is described in many standard laboratory manuals, such as ds In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present 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. It can be recovered from the recombinant cell culture and purified by well-known methods, including chromatography. Most preferably, high performance liquid chromatography ("HPLC")
Is used for purification.

A polypeptide of the invention, and preferably a secreted form, can also be recovered from: purified from natural sources, including body fluids, tissues and cells, whether directly isolated or cultured. The product of the chemical synthesis procedure; and
Products produced by recombinant techniques 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 recombinant production procedures, the polypeptides of the invention may be glycosylated or non-glycosylated. Further, the polypeptides of the present invention may also include, in some cases, an initial modified methionine residue as a result of a host-mediated process. Thus, it is well known in the art that, in general, the N-terminal methionine encoded by the translation initiation codon is removed with high efficiency from any protein after translation in all eukaryotic cells. Although the N-terminal methionine in most proteins is also effectively removed in most prokaryotes, 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 addition to including host cells containing the vector constructs discussed herein, the present invention also relates to primar (primarily mammalian) origin.
y) Including host cells, secondary host cells, and immortalized host cells. These host cells have been engineered to delete or replace endogenous genetic material (eg, a coding sequence) and / or have been genetically operably linked to a polynucleotide of the invention (eg, (A heterologous polynucleotide sequence) and activates, alters, and / or amplifies the endogenous polynucleotide. For example, techniques known in the art can be used to operably link heterologous control regions (eg, promoters and / or enhancers) and endogenous polynucleotide sequences, via homologous recombination, Form a new transfer unit (see, for example, US Pat. No. 5,641,670 issued Jun. 24, 1997; US Pat.
No. 33,761; International Publication No. WO 96/2 published on September 26, 1996
No. 9411; International Publication No. WO 94/1265 published on Aug. 4, 1994
No. 0; Koller et al., Proc. Natl. Acad. Sci. USA 86
: 8932-8935 (1989); and Zijlstra et al., Nature.
342: 435-438 (1989). Each of these disclosures:
Which are incorporated by reference in their entirety).

Further, the polypeptides of the present invention can be chemically synthesized using techniques known in the art (eg, Creighton, 1983, Proteins: Stru.
cultures and Molecular Principles, W.C. H.
Freeman & Co. , N .; Y. And Hunkapiller et al., Na
cure, 310: 105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide sequence of the present invention can be synthesized by use of a peptide synthesizer. In addition, 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, the D isomer of a normal amino acid, 2,4-diaminobutyric acid, a-aminoisobutyric acid,
-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. Further, the amino acid can be D (dextrorotary) or L (levorotary).

The present invention relates to methods for, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, antibody molecules or other cellular activities during or after translation. Includes polypeptides that are differentially modified, such as by binding to a ligand. Any of numerous chemical modifications, including the following may be carried out by known, but not limited to techniques: cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, specific chemical cleavage by 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, the following: N-linked or O-linked carbohydrate chains (N-terminal or C-terminal processing), chemical moieties to the amino acid backbone. Chemical modification of linked, N-linked or O-linked carbohydrate chains, and addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression. The polypeptide may also be modified with a detectable label (eg, an enzymatic, fluorescent, isotopic or affinity label) to allow for 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, such as increased solubility, stability and circulation time, or reduced immunogenicity of the polypeptide. (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 copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, and the like. The polypeptide can be modified at random positions in the molecule or at predetermined positions in the molecule, and can include one, two, three or more attached chemical moieties.

The polymer can be of any molecular weight 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 manufacture (the term "about" means that in the preparation of polyethylene glycol, some molecules may have higher molecular weights than indicated). Heavy and some are lighter than the indicated molecular weight). The desired therapeutic profile (eg, the desired duration of sustained release, in some cases effects on biological activity, ease of handling, degree of antigenicity or lack of antigenicity, and Other sizes may be used, depending on other known effects of polyethylene glycol).

[0437] The polyethylene glycol molecule (or other chemical moiety) should be attached to the protein in view of its effect on the functional or antigenic domains of the protein. There are a number of conjugation methods available to those of skill in the art (see, for example, EP 0 401 384 (P in G-CSF, incorporated herein by reference).
EG), Malik et al., Exp. Hematol. 20: 1028-
1035 (1992) (pegylation of GM-CSF using tresyl chloride)
report)) also see)). For example, polyethylene glycol can be covalently linked via an amino acid residue by a reactive group (eg, a free amino or carboxyl group). Reactive groups are groups to which activated polyethylene glycol molecules can bind. Amino acid residues having a free amino group may 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 can also 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.

A protein chemically modified at the N-terminus may be specifically desired. Using polyethylene glycol as an illustration of the composition of the present invention, the ratio of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mixture from various polyethylene glycol molecules (by molecular weight, branching, etc.), the PEG to be performed. The type of the reaction can be selected and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining an N-terminal PEGylated preparation (ie, if necessary, separating this portion from other mono-PEGylated portions) is accomplished by purification of the N-terminal PEGylated material from a population of PEGylated protein molecules. obtain. Selective proteins chemically modified with N-terminal modification
It can be achieved by reductive alkylation utilizing the differential reactivity of different types of primary amino groups (lysine vs. N-terminal) available for derivatization in certain proteins. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

A polypeptide of the present invention can be a monomer or multimer (ie, dimer,
Trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomeric and multimeric polypeptides of the invention, their preparation and compositions containing them (preferably therapeutics). In certain embodiments, a polypeptide of the invention is a monomer, dimer, trimer or tetramer. In a further embodiment, a multimer of the invention is at least a dimer, at least a trimer, or at least a tetramer.

The multimers encompassed by the present invention can be homomers or heteromers. As used herein, the term homomer refers to polypeptides corresponding to the amino acid sequence of SEQ ID NO: Y or the polypeptide encoded by the cDNA contained in the deposited clone (these polypeptides described herein). (Including fragments, variants, splice variants and fusion proteins corresponding to the peptide). These homomers can include polypeptides having the same or different amino acid sequences. In certain embodiments, a homomer of the invention is a multimer comprising only polypeptides having the same amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer 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 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 a polypeptide of the present invention. In certain embodiments, a multimer of the invention is a heterodimer, heterotrimer or heterotetramer. 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 may be the result of hydrophobic, hydrophilic, ionic and / or covalent associations and / or may be indirectly linked, for example, by liposome formation. Thus, in one embodiment, a multimer of the invention, such as a homodimer or homotrimer, is formed when the polypeptides of the invention contact each other in solution. In another embodiment, the heteromultimers of the invention (
For example, a heterotrimer or heterotetramer) is formed when a polypeptide of the invention is contacted in solution with an antibody to a polypeptide of the invention, including an antibody to a heterologous polypeptide sequence in a fusion protein of the invention. Is done. In other embodiments, multimers of the invention are formed by covalent bonds with and / or between polypeptides of the invention. Such covalent bonds may result in one or more amino acid residues contained in a polypeptide sequence (eg, a polypeptide sequence contained in a polypeptide set forth in the Sequence Listing or encoded by a deposited clone) to be removed. May be included. In one example, this covalent bond is
A bridge between cysteine residues present within a polypeptide sequence that interacts in a native (ie, naturally occurring) polypeptide. In another example, the covalent bond is the result of a chemical or recombinant operation. Alternatively, such covalent bonds may include one or more amino acid residues contained in a heterologous polypeptide sequence in a fusion protein of the invention.

In one example, a covalent bond is between heterologous sequences contained in a fusion protein of the invention (see, eg, US Pat. No. 5,478,925). In certain instances, the covalent bond is between heterologous sequences included in an Fc fusion protein of the invention (as described herein). In another specific example, the covalent linkage of a fusion protein of the invention is between heterologous polypeptide sequences from another protein (eg, such as osteoprotegerin) that can form a covalently linked multimer (eg, See International Publication No. WO 98/49305, the contents of which are incorporated herein by reference in its 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, which is incorporated herein by reference. Proteins comprising a plurality of polypeptides of the invention separated by a peptide linker 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 or isoleucine zipper polypeptide sequence. Leucine zipper domains and isoleucine zipper domains are polypeptides that promote multimerization of the protein in which the domain is found. Leucine zippers originally had several DNs
A-binding protein (Landschulz et al., Science
240: 1759, (1988)), and since then have been found in a variety of different proteins. In particular, the known leucine zippers are naturally occurring peptides and derivatives thereof that form dimers or trimers. Examples of suitable leucine zipper domains for producing 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. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimers in solution can be expressed in a suitable host cell, and the resulting soluble multimeric fusion protein can be expressed in the art. And recovered from the culture supernatant using a known technique.

[0445] 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 Ho, which is incorporated herein by reference.
Leucine zippers from pulmonary surfactant protein D (SPD) as described in ppe et al. (FEBS Letters 344: 191, (1994)) and U.S. 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 invention.

In another example, the proteins of the invention associate by interaction between Flag® polypeptide sequences contained in a fusion protein of the invention that includes the Flag® polypeptide sequence. In a further embodiment, the association of a protein of the invention is associated by an interaction between a heterologous polypeptide sequence contained in a Flag® fusion protein of the invention and an anti-Flag® antibody.

The multimers of the present invention can be produced using chemical techniques known in the art. For example, a polypeptide that is desired to be included in a multimer of the present invention can be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (eg, as described herein). See U.S. Patent No. 5,478,925, which is incorporated by reference in its entirety). In addition, the multimers of the present invention may be generated using techniques known in the art to reduce one or more intermolecular cross-links between cysteine residues present within the sequence of the polypeptide desired to be included in the multimer. (See, for example, US Pat.
478,925). Furthermore, the polypeptides of the present invention can be conventionally modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide, and techniques known in the art may be used to modify one or more of these modified poly- It can be applied to generate multimers containing peptides (see, eg, US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety). In addition, techniques known in the art can be applied to generate liposomes containing the polypeptide components desired to be included in the multimers of the invention (eg, incorporated herein by reference in their entirety). US Patent No. 5,4
78, 925).

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

Use of Polynucleotides Each of the polynucleotides identified herein can be used in a number of ways as reagents. The following description is to be regarded as illustrative and utilizes known techniques.

[0450] The polynucleotides of the present invention are useful for chromosome identification. Since chromosome marking reagents based on actual sequence data (repetitive polymorphisms) are currently scarcely available, there remains a need to identify new chromosomal markers. Each polynucleotide of the present invention can be used as a chromosome marker.

[0451] Briefly, sequences can be mapped to chromosomes 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 to identify the P of somatic cell hybrids containing individual human chromosomes.
Used for CR screening. Only those hybrids containing the human gene corresponding to SEQ ID NO: X will produce an amplified fragment.

[0452] Similarly, somatic cell hybrids have the ability to target polynucleotides to specific chromosomes.
Provides a quick method for CR mapping. More than two clones can be assigned per day using a single thermal cycler. In addition, sublocalization of a polynucleotide can be accomplished using a panel of specific chromosomal fragments. Other gene mapping strategies that can be used include in situ hybridization, labeled flow sorting (labeled
flow sorted) chromosomes and preselection by hybridization to construct a chromosome-specific cDNA library.

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

For chromosome mapping, polynucleotides may be used individually (to mark a single chromosome or a single site on that chromosome) or in a panel (to mark multiple sites and / or multiple chromosomes). Can be done. Preferred polynucleotides correspond to non-coding regions of the cDNA. Because the coding sequences tend to be more conserved within the gene family, this increases the chance of cross-hybridization during chromosome mapping.

Once the polynucleotide has been mapped to a precise chromosomal location, the physical location of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coherence between chromosomal location and presentation of a particular disease. (Disease mapping data is described, for example, in 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 correctly located in a chromosomal region associated with a 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 tested in chromosome spreads or by PCR. The absence of a structural change confirms the presence of a point mutation. Mutations observed in some or all affected individuals, but not in normal individuals, indicate 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, the polymorphic polypeptide can be used for further linkage analysis.

In addition, increased or decreased expression of a gene in an affected individual as compared to an unaffected individual can be assessed using a polynucleotide 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 diagnostic method useful during the diagnosis of a disorder, comprising the step of measuring the expression level of a polynucleotide of the present invention in cells or body fluids from an individual; Comparing the measured gene expression level to a standard level of polynucleotide expression level, whereby an increase or decrease in gene expression level relative to the standard is indicative of the disorder.

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

If the diagnosis of the 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 are closer to standard levels Experience poor clinical results as compared to patients expressing this gene at the level.

By “measuring the expression level of a polynucleotide of the present invention”, the level of the polypeptide of the present invention or the level of the mRNA encoding this polypeptide can be measured directly in the first biological sample. (E.g., by determining or evaluating absolute protein or mRNA levels) or relative (
(Eg, by comparing against polypeptide or mRNA levels in a second biological sample), either qualitatively or quantitatively. Preferably, the polypeptide or mRNA level in the first biological sample is measured or evaluated and compared to a standard polypeptide or mRNA level, wherein the standard is obtained from an individual without a disorder. Determined by averaging levels obtained from a second biological sample obtained or from a population of individuals without the disorder. As will be recognized in the art, once a standard polypeptide 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, a body fluid, a cell line, a tissue culture or other source that contains a polypeptide or mRNA of the present invention. As shown, biological samples may express body fluids (eg, semen, lymph, serum, plasma, urine, synovial fluid and cerebrospinal fluid) containing polypeptides of the invention, and polypeptides 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, a tissue biopsy is the preferred source.

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

The present invention encompasses polynucleotides of the present invention that have been 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 DNA analogs of the polyamide type, and the monomer units for adenine, guanine, thymine and cytosine are commercially available (Perceptice Biosystems). DNA
Specific components (eg, phosphorus, phosphorus oxide or deoxyribose derivatives)
Not present in NA. P. E. FIG. Nielsen, M .; Egholm, R .; H. B
erg and O. erg. Buchardt, Science 254, 1497 (19
97); Egholm, O.M. Buchardt, L .; Christ
ensen, C.I. Behrens, S .; M. Freier, D .; A. Drive
r, R. H. Berg, S.M. K. Kim, B .; Norden and P.M. E. FIG. Ni
As disclosed by Elsen, Nature 365,666 (1993), PNA binds specifically and tightly to complementary DNA strands and is not degraded by nucleases. In fact, PNA binds DNA more strongly than DNA itself. This is probably due to the absence of electrostatic repulsion between the two chains and also the more flexible polyamide backbone. by this,
PNA / DNA duplexes bind under a wider range of stringency conditions than DNA / DNA duplexes, making it easier to perform multi-stranded hybridization. Due to the strong binding, smaller probes can be used than with DNA. Further, perhaps a single base mismatch can be determined using PNA / DNA hybridization. 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.
On the other hand, the melting point (T.sub.m) is reduced by 8 to 20 ° C. 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 detecting cancer in mammals. In particular, the invention is useful during the diagnosis of pathological cell proliferative 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, etc.); and chronic myelogenous leukemia (chronic myelomonocytic leukemia, chronic granulocytic Leukemia, etc.). Preferred mammals include monkeys, monkeys, ape, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.

[0466] Pathological cell proliferative disorders are often associated with inappropriate activation of proto-oncogenes (
Gelmann, E .; P. Et al., "The Etiology of Accute."
Leukemia: Molecular Genetics and Vir
al Oncology ", Neoplastic Diseases of
the Blood, Volume 1, Wiernik, P .; H. Ed., 161-182
(1985)). Neoplasia is currently caused by the insertion of viral sequences into the chromosome, chromosomal translocation of the gene to a more actively transcribed region, or by some other mechanism, from qualitative changes in normal cellular gene products, or from gene expression It is believed to arise from quantitative modifications (Gelmann et al., Supra). Mutations or altered expression of certain genes appear to be involved in the pathogenesis of some leukemias, among other tissues and other cell types (Gelmann et al., Supra). Indeed, human counterparts of some oncogenes involved in animal neoplasia have been amplified or translocated in some cases of human leukemia and cancer (Gelmann et al., Supra). For example, c-my
c expression is highly amplified in the non-lymphocytic leukemia cell line HL-60. H
When L-60 cells are chemically induced to stop amplification, levels of c-myc are found to be down-regulated (International Publication No. WO 91/1).
No. 5580). However, exposure of HL-60 cells to a DNA construct that is complementary to the 5 ′ end of c-myc or c-myb results in a decrease in c-myc protein or cm
It has been shown to block translation of the corresponding mRNA that down-regulates expression of the yb protein, causing cessation of cell growth and differentiation of treated cells (WO 91/15580; Wickstrom et al., Proc.
. Natl. Acad. Sci. 85: 1028 (1988); Anfossi.
Proc. Natl. Acad. Sci. 86: 3379 (1989)).
However, one of skill in the art would recognize that the utility of the present invention would be useful in treating proliferative disorders of hematopoietic cells and tissues, given the large number of cells and cell types of various origins known to exhibit a proliferative phenotype. Recognize that it is not limited.

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 .; 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 the binding of the polynucleotide to complementary DNA or complementary RNA. For these techniques, preferred polynucleotides are usually oligonucleotides of 20 to 40 bases in length, and the 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 the 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 the blockade of RNA transcription from DNA, whereas antisense RNA hybridization prevents translation of the mRNA molecule into a polypeptide. 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 disease.

[0468] The polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism that has the defective gene in an attempt to correct the genetic defect. The polynucleotide disclosed in the present invention,
It provides a means to target such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby creating a new trait in the host cell.

[0469] Polynucleotides are also useful for identifying individuals from small biological samples. For example, the United States military is considering the use of restriction fragment length polymorphisms (RFLPs) to identify its personnel. In this technique, an individual's genome D
NA is digested with one or more restriction enzymes and probed on a Southern blot 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, replaced, or stolen.
The polynucleotide of the present invention can be used as an additional DNA marker for RFLP.

The polynucleotides of the present invention can also be used as an alternative to RFLP by determining the actual base-by-base DNA sequence of 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 the selected DNA can then be sequenced. Using this technique, individuals can be identified as each individual has a unique set of DNA sequences. Once a unique ID database has been established for an individual, whether the individual is alive or dead, positive identification of the individual can be made from very small tissue samples.

Forensic biology may also benefit 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 surfactants, urine, fecal material, etc.) can be amplified using PCR.
In certain prior art, gene sequences amplified from polymorphic loci such as the DQa class II HLA gene are used in forensic biology to identify individuals. (Errich, H., PCR Technology, Freem.
and and Co. (1992)). Once these specific polymorphic loci have been amplified, they are digested with one or more restriction enzymes. This results in an identified set of bands for Southern blots probed with DNA corresponding to the DQa class II HLA gene. Similarly, the polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.

There is also a need for reagents that can identify specific tissue sources. Such a need arises in forensic medicine, for example, when tissue of unknown origin is provided. Suitable reagents can include, for example, DNA probes or primers specific to a particular tissue, prepared from the sequences of the present invention. Such a panel of reagents may identify tissue 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 “subtract known sequences in the process of discovering novel polynucleotides, as molecular weight markers in Southern gels, as diagnostic probes for the presence of particular mRNAs in particular cell types. To select and produce oligomers for attachment to a "gene chip" or other support, to elicit anti-DNA antibodies using DNA immunization techniques, and to elicit an immune response. Can be used as

Use of Polypeptides Each of the polypeptides identified herein can be used in many ways. The following description is to be regarded as illustrative and utilizes known techniques.

The polypeptides of the present invention can be used to assay protein levels in biological samples using antibody-based techniques. For example, protein expression in tissues can be studied using classical immunohistochemistry (Jalka
nen, M .; J. et al. Cell. Biol. 101: 976-185 (1985)
Jalkanen, M .; J. 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 assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include, for example, enzyme labels such as glucose oxidase, and iodine (125I, 121I), carbon (14I).
C), radioactive isotopes such as sulfur (35S), tritium (3H), indium (112In), and 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 radioisotopes such as barium or cesium which 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 relevant hybridomas .

Protein-specific, 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 antibody or antibody fragment is introduced into a mammal (eg, parenterally, subcutaneously, or intraperitoneally). It is understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety required to generate a diagnostic image. In the case of a radioisotope moiety,
For human subjects, the amount of radioactivity injected is typically about 5-20 μm of 99mTc.
Millicurie range. The labeled antibody or antibody fragment is then preferentially accumulated at locations in the cell that contain the particular protein. In vivo tumor imaging is described by S.D. W. Burchiel et al., "Immunopharmacokine."
tics of Radiolabeled Antibodies and
Their Fragments. (Tumor Imaging: The
Radiochemical Detection of Cancer No.1
Chapter 3, S.M. W. Burchiel and B.A. A. Rhodes, Masson
Publishing Inc. (1982)).

Accordingly, the present invention provides a method of diagnosing a disorder, comprising: (a) assaying the expression of a polypeptide of the present invention in cells or body fluids of an individual; (b) the level of gene expression Comparing to a standard gene expression level, whereby an increase or decrease in the gene expression level of the assayed polypeptide relative to the standard expression level is indicative of the disorder. For cancer, can the presence of relatively large amounts of transcripts in biopsy tissue from an individual indicate a predisposition to the development of the disease,
Or it may provide a means to detect this disease before the actual clinical symptoms appear.
A more conclusive diagnosis of this type may allow health professionals to use prophylactic measures or aggressive treatment earlier, thereby preventing the onset or further progression of cancer.

Further, a disease of the present invention can be treated using the polypeptide of the present invention. For example, a patient may be able to replace the absence or reduced level of a polypeptide (eg, insulin), replace a different polypeptide (eg, hemoglobin S versus hemoglobin B,
Supplementing the absence or reduced level of SOD, catalase, DNA repair protein), inhibiting the activity of a polypeptide (eg, an oncogene or a tumor suppressor), binding the activity of the polypeptide (eg, to a receptor) Activating), reducing the activity of the membrane-bound receptor by competing with the membrane-bound receptor for free ligand (eg, soluble TNF receptor used in reducing inflammation), or the desired response ( For example, a polypeptide of the invention may be administered in addressing its effects (eg, inhibiting growth of blood vessels, enhancing the immune response to proliferating cells or tissues).

Similarly, antibodies to a polypeptide of the invention may also be used to treat a disease. For example, administration of an antibody to a polypeptide of the invention can bind to the polypeptide and reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate that polypeptide, for example, by binding to a membrane-bound polypeptide (receptor).

At least the polypeptides of the present invention may be prepared by SDS-P using methods well known to those skilled in the art.
It can be used as a molecular weight marker for AGE gel 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 way to assess 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 present invention relates to a gene therapy method for treating disorders, diseases, and conditions. This method of gene therapy involves introducing nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to achieve expression of a polypeptide of the invention. This method requires a polynucleotide encoding a polypeptide of the present invention operably linked to a promoter and any other genetic elements required for expression of the polypeptide by the target tissue. Techniques for such gene therapy and delivery are known in the art, see, for example, WO 90/11092, which is incorporated herein by reference.

Thus, for example, a cell from a patient may be 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 a 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. 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 cells to be manipulated are arterial cells. The arterial cells can be reintroduced into the patient by direct injection into the artery, peri-arterial tissue, or by catheter injection.

As discussed in more detail below, the polynucleotide constructs can be used to deliver any injectable substance to the cells of an animal, including, but not limited to, tissues (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, a polynucleotide of the invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA, refers to any delivery vehicle (eg, that acts to assist, facilitate or facilitate entry into cells).
Virus sequences, virus particles, liposome formulations, lipofectin, 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 U.S. Pat. Nos. 5,593,972, 5,589, incorporated herein by reference.
, 466 and 5,580,859.

The polynucleotide vector constructs of the present invention for use in gene therapy methods are preferably not integrated into the host genome and contain no sequences enabling replication.
It 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 one skilled in the art.

[0487] Any strong promoter known to those of skill in the art can be used to drive expression of a polynucleotide sequence of the present invention. Suitable promoters include an adenovirus promoter (eg, the adenovirus major late promoter); or a heterologous promoter (eg, the cytomegalovirus (CMV) promoter);
RS virus (RSV) promoter; inducible promoters (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); LTR; b-actin promoter; and human growth hormone promoter. This promoter can also be a native promoter for the polynucleotide of the present invention.

[0488] Unlike other gene therapy techniques, one major advantage of introducing a naked nucleic acid sequence into a target cell is the transient nature of polynucleotide synthesis in that cell. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

The polynucleotide constructs of the present invention can be used in tissues (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, eyes, glands, and connective tissue)
To the interstitial space. The interstitial space of the tissue is between the cells between the reticulum fibers of the organ tissue, the mucopolysaccharide matrix of the fluid, the elastic fibers in the walls of the blood vessels or chambers, the collagen fibers in the fibrous tissue, the connective tissue muscular cells or the bones. Includes the same substrate in the cleft. This is also the space occupied by the circulating plasma and the lymph of the lymph channels. Delivery of muscle tissue to the interstitial space is preferred for reasons discussed below. They can be conveniently delivered by injection into the tissue containing these cells. They are preferably delivered to and expressed in differentiated, persistent, non-dividing cells, but the delivery and expression can be in undifferentiated cells or in fully differentiated cells (eg, blood stem cells or Skin fibroblasts). In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

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

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

[0492] Naked polynucleotide may be administered by direct needle injection, intravenous injection, topical administration at the site of delivery,
Including, but not limited to, catheter injection, and so-called "gene guns";
It is delivered by any method known in the art. These delivery methods are known in the art.

The construct can also be delivered using a delivery vehicle such as a viral sequence, viral particles, liposome formulation, lipofectin, precipitant, and the like. Such delivery methods are known in the art.

In certain embodiments, a polynucleotide construct of the invention is complexed in a liposome preparation. Preparations of liposomes 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 tightly charged complex between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes, in their functional form, contain plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. U, incorporated herein by reference).
SA, 84: 7413-7416 (1987)); mRNA (Malone et al., Proc. Natl. Acad. Sci., Incorporated herein by reference).
ScL USA, 86: 6077-6081 (1989)); and purified transcription factors (Debs et al., J. Biol. Chem., Incorporated herein by reference).
265: 10189-10192 (1990)).

[0495] 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 available under the trademark Lipofectin from GIBCO BRL, Grand Island, N.B. Y. (Felgner et al., Proc. Natl. Acad. Sc, which is incorporated herein by reference.
i. USA, 84: 7413-7416 (1987)). Other commercially available liposomes include transfectase (tr
anffect) (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, for example, PCT Publication No. WO 90/11092, which is incorporated herein by reference. The preparation of DOTMA liposomes has been described in the literature and is described, for example, in Felgner et al., Proc. Na
tl. Acad. Sci. ScL USA, 84: 7413-7417. Similar methods can be used to prepare liposomes from other cationic lipid substances.

Similarly, anionic and neutral liposomes are available from Avanti Po.
It is readily available from sources such as lar Lipids (Birmingham, Ala.) or can be easily prepared using readily available materials. Such substances include, inter alia, phosphatidyl, choline, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE). These materials can also be mixed in appropriate proportions with the DOTMA and DOTAP starting materials. Methods for producing liposomes using these materials are well known in the art.

For example, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) are used commercially in various combinations with and without the addition of cholesterol. Thus, conventional liposomes can be produced. Thus, for example, in a sonicated vial under a nitrogen gas flow, DOPG and DOPC each 50 m
By drying the g, DOPG / DOPC vesicles can be prepared. The sample is placed under a vacuum pump overnight and hydrated the next day with deionized water. Then, using a Heat Systems Model 350 sonicator equipped with an inverted cup (bath type) probe at maximum settings while the bath is circulating at 15EC.
The sample is sonicated for 2 hours in a stoppered vial. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles, or to generate discrete sized unilamellar vesicles by extrusion through a nuclear pore membrane. I can do it. Other methods are known and available to those skilled in the art.

The liposomes include multilamellar vesicles (MLV), small unilamellar vesicles (SUV)
Or large unilamellar vesicles (LUV) may be mentioned, with SUV being preferred. Various liposome-nucleic acid complexes are prepared using methods well known in the art. For example,
Straubinger et al., Method, incorporated herein by reference.
s of Immunology, 101: 512-527 (1983). For example, MLVs containing nucleic acids can be prepared by depositing a thin film of phospholipid on the wall of a glass tube, followed by hydration with a solution of the substance to be encapsulated. SUVs are prepared by prolonged sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be encapsulated is added to a pre-formed suspension of MLV and then sonicated. When using liposomes containing cationic lipids, the dried lipid membrane is resuspended in a suitable solution such as sterile water or an isotonic buffer solution such as 10 mM Tris / NaCl, sonicated, and then sonicated. ,
The preformed liposomes are mixed directly with the DNA. Due to the binding of positively charged liposomes to cationic DNA, liposomes and DNA form very stable complexes. SUV finds use for small nucleic acid fragments. LU
V is prepared by a number of methods well known in the art. Commonly used methods include Ca ²⁺ -EDTA chelation (Papahadjopoulos et al., B
iochim. Biophys. Acta, 394: 483 (1975); Wi.
lson et al., Cell, 17:77 (1979)); ether injection (Deame).
r et al., Biochim. Biophys. Acta, 443: 629 (1976).
Ostro et al., Biochem. Biophys. Res. Comum.
76: 836 (1977); Fraley et al., Proc. Natl. Acad
. Sci. USA, 76: 3348 (1979)); Surfactant dialysis (Enoc
h et al., Proc. Natl. Acad. Sci. USA, 76: 145 (197
9)); and reversed-phase evaporation (REV) (Fraley et al., J. Bio.)
l. Chem. 255: 10431 (1980); Szoka et al., Proc.
Natl. Acad. Sci. USA, 75: 145 (1978); Schae.
fer-Rider et al., Science, 215: 166 (1982)), which are incorporated herein by reference.

Generally, the ratio of DNA to liposomes 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.

[0501] US Patent No. 5,676,954, incorporated herein by reference, reports the injection of genetic material conjugated with a cationic liposome carrier into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, and 5,589,466.
No. 5,693,622, No. 5,580,859, No. 5,703
No. 055 and WO 94/9469, which are incorporated herein by reference, provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. No. 5,589,466;
Nos. 5,693,622, 5,580,859 and 5,703,05
No. 5 and WO 94/9469, which are incorporated herein by reference, provide methods for delivering DNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, using retroviral particles comprising RNA containing a sequence encoding a polypeptide of the invention. Retroviruses that can induce a retroviral plasmid vector include Moloney mouse leukemia virus, spleen necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, gibbon leukemia virus,
Examples include, but are not limited to, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary carcinoma virus.

The retroviral plasmid vector is used to transduce a packaging cell line to form a producer cell line. Examples of packaging cell lines that can be transfected include M, M, which is incorporated herein by reference in its entirety.
iller, Human Gene Therapy, 1: 5-14 (1990).
PE501, PA317, R-2, R-AM, PA12, as described in
T19-14X, VT-19-17-H2, RCRE, RCRIP, GP + E-
86, GP + envAm12 and the DAN cell line. This vector can transduce the packaging cells by any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, CaPO ₄ precipitation. In one alternative, the retroviral plasmid vector can be encapsulated in liposomes or conjugated to a lipid and then administered to a host.

The producer cell line produces infectious retroviral vector particles that contain 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. The transduced eukaryotic cells will express the polypeptide of the invention.

In certain other embodiments, cells are engineered, ex vivo or in vivo, with a polynucleotide of the invention contained in an adenovirus vector. An adenovirus can be engineered such that it encodes and expresses a polypeptide of the present invention, while at the same time being inactivated with respect to its ability to replicate in the normal lytic viral life cycle. Adenovirus expression is achieved without the integration of viral DNA into the host cell chromosome, thereby reducing concerns about insertional mutagenesis. In addition, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz et al., Am. Rev.
Respir. Dis. , 109: 233-238 (1974)). Finally,
Adenovirus-mediated gene transfer comprises α-1-antitrypsin and CFTR
Has been demonstrated in a number of cases, including transfer into the lungs of cotton rats (Rose
nfeld et al., Science, 252: 431-434 (1991); Ros.
enfeld et al., Cell, 68: 143-155 (1992)). In addition, a great deal of research trying to establish adenovirus as a causative agent in human cancer has
It was uniformly negative (Green et al. Proc. Natl. Acad. S.
ci. ScL USA, 76: 6606 (1979)).

Suitable adenoviral vectors useful in the present invention are, for example, Kozar
sky and Wilson, Curr. Opin. Genet. Devel. ,
3: 499-503 (1993); Rosenfeld et al., Cell, 68: 1.
43-155 (1992); Engelhardt et al., Human Genet.
. Ther. 4: 759-769 (1993); Yang et al., Nature.
Genet, 7: 362-369 (1994); Wilson et al., Nature.
365: 691-692 (1993); and U.S. Patent No. 5,652,224.
And these are incorporated herein by reference. For example, the adenovirus vector Ad2 is useful and can be propagated in human 293 cells. These cells contain the E1 region of the adenovirus and constitutively express Ela and Elb, which complement the defective adenovirus by providing the product of the gene that is deleted from the 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 virus and / or packaging cell lines to form infectious particles. The resulting virus is capable of infecting cells and can express the polynucleotide of interest linked to a promoter, but cannot replicate in most cells. The replication defective adenovirus can be deleted in all or part of one or more of the following genes: E1a, E1b, E3, E4, E2a or L1 to L5.

In certain other embodiments, cells are engineered ex vivo or in vivo using an adeno-associated virus (AAV). AAV is a naturally occurring defective virus that requires a helper virus to produce infectious particles (Muz
yczka, Curr. Topics in Microbiol. Immun
ol. , 158: 97 (1992)). It is also one of the few viruses that can integrate its DNA into non-dividing cells. Vectors containing AAV as small as 300 base pairs can be packaged and integrated,
Space for exogenous DNA is limited to about 4.5 kb. Methods for making and using such AAV are known in the art. See, for example, US Pat.
No. 5,941, No. 5,173,414, No. 5,354,678, No. 5,
See 436,146, 5,474,935, 5,478,745 and 5,589,377.

For example, AAV vectors suitable for use in the present invention include all necessary sequences for DNA replication, encapsidation, and host cell integration. Sa
mbrook et al., Molecular Cloning: A Laboratory.
ry Manual, Cold Spring Harbor Press (1
998) using standard cloning methods, such as the method found in
A polynucleotide construct comprising a polynucleotide of the invention is inserted into an AAV vector. The recombinant AAV vector is then transfected into packaging cells infected with the 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 have been transfected and infected, they produce infectious AAV virions containing a polynucleotide construct of the invention. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cell contains the polynucleotide construct integrated into its genome, and expresses the desired gene product.

Another method of gene therapy uses homologous recombination (eg, US Pat. No. 5,641,6).
No. 70, issued on June 24, 1997; International Publication No. WO 96/29411, 19
Published September 26, 1996; International Publication No. WO 94/12650, August 4, 1994
Koller et al., Proc. Natl. Acad. Sci. USA, 8
6: 8932-8935 (1989); and Zijlstra et al., Natur.
e, 342: 435-438 (1989)). including. This method involves the activation of a gene that is present in the target cell, but is not normally expressed in that cell, or that is expressed at a lower level than desired.

[0511] Polynucleotide constructs are made using standard techniques known in the art. The construct includes the promoter with a target sequence adjacent to the promoter.
Suitable promoters are described herein. The target sequence is sufficiently complementary to the endogenous sequence to allow for homologous recombination between the promoter target sequence and the endogenous sequence. The target sequence is sufficiently close to the 5 'end of the desired endogenous polynucleotide sequence, and thus, by homologous recombination, the promoter is operably linked to the endogenous sequence.

[0512] The promoter and target sequence can be amplified using PCR. Preferably, the propagated promoter contains different restriction sites at the 5 'and 3' ends. Preferably, the 3 'end of the first target sequence contains the same restriction sites as the 5' end of the amplified promoter, and the 5 'end of the second target sequence has the same restriction sites as the 3' end of the amplified promoter. Contains enzyme sites. The amplified promoter and target sequence are digested and ligated together.

As a naked polynucleotide or together with transfection enhancing agents such as liposomes, viral sequences, viral particles, whole viruses, lipofections, precipitants, etc., as described in more detail above. Deliver the promoter-target sequence construct to the cell. The P promoter-target sequence can be delivered by any method, including direct needle injection, intravenous injection, local administration, catheter infusion, particle accelerator, and the like. This method is described in more detail below.

[0514] The promoter-target sequence construct is taken up by the cell. Homologous recombination between the construct and the endogenous sequence occurs, such that the endogenous sequence is placed under the control of the promoter. This promoter then drives the expression of the endogenous sequence.

[0515] Polynucleotides encoding the polypeptides of the present invention can be administered together with other polynucleotides encoding other angiogenic proteins. Angiogenic proteins include acidic and basic fibroblast growth factors, VEGF-1, VEG
F-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factors 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 nitric oxide synthase.

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

According to the method, any method of administration of any of the polynucleotide constructs described above can be used, so long as the one or more molecules are expressed 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"), gel foam sponge depots,
Other commercially available depot materials, including osmotic pumps (eg, Alza minipumps), oral or suppository solid (tablets or pills) pharmaceutical formulations, and intraoperative decanting or topical application. For example, direct injection of calcium phosphate-precipitated naked plasmid into rat liver and rat spleen or direct injection of protein-coated plasmid into portal vein resulted in gene expression of exogenous genes in rat liver (Ka
neda et al., Science, 243: 375 (1989)).

[0518] A preferred method of topical administration is by direct injection. Preferably, the recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into the arterial region or locally within the arterial region. Local administration of the composition within the area of the artery refers to injecting the composition into the artery a few centimeters, preferably a few millimeters.

Another method of local administration is to contact the polynucleotide construct of the present invention in or around a surgical wound. For example, a patient may undergo surgery and may coat the polynucleotide construct on a tissue surface inside the wound, or may inject the construct into a tissue area inside the wound.

[0520] Therapeutic compositions useful for systemic administration comprise a recombinant molecule of the present invention complexed with a targeted delivery vehicle of the present invention. Delivery vehicles suitable for use in systemic administration include liposomes that contain a ligand that targets the vehicle to a particular site.

[0521] Preferred methods of systemic administration include intravenous injection, aerosol, oral and transdermal (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery may also be performed using methods standard in the art (eg, Stribling et al., Proc. Natl. Acad. Sci. USA, 189: 11277-11, incorporated herein by reference).
281 (1992)). Oral delivery can be performed by forming a complex of the polynucleotide construct of the present invention with a carrier capable of withstanding degradation by digestive enzymes in the intestine of the animal. Examples of such carriers include plastic capsules or tablets, such as those known in the art. Topical delivery can be accomplished by mixing a polynucleotide construct of the invention with a lipophilic reagent (eg, DMSO) that can pass into the skin.

Determining the 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 precise condition requiring treatment and its severity, and the route of administration May depend on a number of factors, including The frequency of treatment will depend 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 a physician or veterinarian. The therapeutic compositions of the present invention can be administered to any animal, preferably mammals and birds. Preferably, 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 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, it is likely that these molecules can be involved in diseases associated with their biological activity. Thus, polynucleotides or polypeptides, or agonists or antagonists, can be used to treat the associated disease.

(Immunological activity) The polynucleotide or polypeptide, or agonist or antagonist of the present invention activates or inhibits the proliferation, differentiation, or recruitment (chemotaxis) of immune cells to thereby cause a deficiency in the immune system or It may be useful in treating disorders. Immune cells develop through a process called hematopoiesis, producing myeloid cells (platelets, red blood cells, neutrophils and macrophages) and lymphoid cells (B and T lymphocytes) from pluripotent stem cells. The etiology of these immune deficiencies or disorders can be genetic, somatic (eg, cancer or some autoimmune disorders), acquired (eg, due to chemotherapy or toxins) or infectious. further,
The polynucleotide or polypeptide, or agonist or antagonist, of the present invention may be a marker or detectable substance (de) for a particular immune system disease or disorder.
Tector).

The polynucleotides or polypeptides, or agonists or antagonists, of the present invention may be useful in treating or detecting hematopoietic cell deficiencies and disorders. The polynucleotides or polypeptides, or agonists or antagonists of the invention, may be used to differentiate and proliferate hematopoietic cells, including pluripotent stem cells, in an attempt to treat disorders associated with the reduction of certain (or many) types of hematopoietic cells. Can be used to increase the Examples of immunological deficiency syndromes include blood protein disorders (eg, agammaglobulinemia, hypogammaglobulinemia),
Ataxia telangiectasia, unclassified immunodeficiency, DiGeorge syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, bactericidal bactericidal dysfunction, severe combined immunodeficiency (SCID), But not limited to, Viscott-Aldrich disorder, anemia, thrombocytopenia, or hemoglobinuria.

In addition, polynucleotides or polypeptides of the invention, or agonists or antagonists, may also be used to modulate hemostatic activity (stop bleeding) or thrombolytic activity (clotting). For example, by using the polynucleotide or polypeptide of the present invention or an agonist or antagonist, a blood coagulation disorder (eg, fibrinogenesis, factor deficiency), blood platelet disorder can be obtained by increasing hemostatic activity or thrombolytic activity. (Eg, thrombocytopenia), or wounds resulting from trauma, surgery or other causes. Alternatively, a clot may be inhibited or lysed using a polynucleotide or polypeptide of the invention, or an agonist or antagonist, capable of reducing hemostatic or thrombolytic activity.
These molecules may be important in treating heart attacks (infarctions), strokes or scars.

In treating or detecting an autoimmune disorder, the polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be useful. Many autoimmune disorders result from improper self-recognition by immune cells as a foreign substance. This inappropriate recognition triggers an immune response that results in the destruction of the host tissue. Thus, administration of a polynucleotide or polypeptide of the invention, or an agonist or antagonist, that inhibits an immune response, particularly T cell proliferation, differentiation or chemotaxis, may be an effective treatment in preventing an autoimmune disorder.

Examples of autoimmune disorders that can be treated or detected by the present invention include: Addison's disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture Syndrome, Graves' disease, multiple sclerosis, myasthenia gravis, neuritis, ophthalmic pain, bullous pemphigoid, pemphigus, polyendocrine disease, purpura, Reiter's disease, Stiffman syndrome, autoimmunity These include, but are not limited to, thyroiditis, systemic lupus erythematosus, autoimmune pulmonary inflammation, Guillain-Barre syndrome, insulin-dependent diabetes, and autoimmune inflammatory eye diseases.

Similarly, such as asthma (particularly allergic asthma) or other respiratory problems,
Allergic reactions and conditions can also be treated with a polynucleotide or polypeptide of the present invention, or with an agonist or antagonist. In addition, these molecules can be used to treat anaphylaxis, hypersensitivity or blood group incompatibility to antigenic molecules.

The polynucleotides or polypeptides of the invention, or agonists or antagonists, may also be used to treat and / or prevent organ rejection or host versus graft disease (GVHD). Organ rejection results from the destruction of transplanted tissue by host immune cells through an immune response. Similarly, the immune response is also involved in GVHD, where exogenous transplanted immune cells destroy host tissues. Administration of a polynucleotide or polypeptide, or agonist or antagonist of the invention, that inhibits the immune response, particularly T cell proliferation, differentiation or chemotaxis, may be an effective treatment in preventing organ rejection or GVHD.

Similarly, polynucleotides or polypeptides of the present invention, or agonists or antagonists, may also be used to modulate inflammation. For example, the polypeptide or polynucleotide, or agonist or antagonist, can inhibit the growth and differentiation of cells involved in the inflammatory response. Using these molecules, infections (eg, septic shock, sepsis, or systemic inflammatory response syndrome (S
IRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disorders, Crohn's disease or cytokines (eg, TNF or IL) It can treat both chronic and acute inflammatory conditions, including those associated with those resulting from overproduction of -1).

Hyperproliferative Disorders The polynucleotides or polypeptides of the invention, or agonists or antagonists, can be used to treat or detect hyperproliferative disorders, including neoplasms.
A polynucleotide or polypeptide, or agonist or antagonist, of the present invention may inhibit the growth of this disorder by direct or indirect interactions. Alternatively, the polynucleotides or polypeptides, or agonists or antagonists of the invention, can proliferate other cells that can inhibit hyperproliferative disorders.

For example, a hyperproliferative disorder can be treated by increasing the immune response, particularly by increasing the antigenic quality of the hyperproliferative disorder, or by expanding, differentiating, or recruiting T cells. This immune response can be increased either by enhancing the existing immune response or initiating a new immune response. Alternatively, reducing the immune response, such as a chemotherapeutic agent, may also be a method of treating a hyperproliferative disorder.

Examples of hyperproliferative disorders that can be treated or detected by the polynucleotides or polypeptides of the invention, or agonists or antagonists, include abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, Parathyroid, pituitary,
Testes, ovaries, thymus, thyroid), eyes, head and neck, nerves (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, rib cage, and neoplasms located in the genitourin It is not limited to.

Similarly, other hyperproliferative disorders can also be treated or detected by the polynucleotides or polypeptides of the invention, or agonists or antagonists. Examples of such hyperproliferative disorders include hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemia, purpura, sarcoidosis, Sezary syndrome, Waldenstrom's macroglobulinemia, Goshe disease, histiocytosis Proliferative diseases and any other hyperproliferative diseases, as well as neoplasms located in the organ systems listed above.

One preferred embodiment utilizes the polynucleotides of the present invention,
And / or gene therapy using a protein fusion or fragment thereof inhibits abnormal cell division.

Thus, the present invention provides a method for treating a cell proliferative disorder by inserting a polynucleotide of the present invention into an abnormally growing cell. Here, the above-mentioned polynucleotide suppresses the above-mentioned expression.

[0538] Another embodiment of the present invention provides a method of treating a cell proliferative disorder in an individual, the method comprising administering one or more active gene copies of the present invention to abnormally proliferating cells. The step of performing 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 polynucleotide described above. In another preferred embodiment of the invention, a DNA construct encoding a polynucleotide of the invention may be inserted into a cell and treated utilizing a retrovirus (or more preferably, an adenovirus vector) (as a reference). Incorporated, GJ.Nabel et al., PNAS.
1999 96: 324-326). In a most preferred embodiment, the viral vector is defective and does not transform non-proliferating cells, but only transforms proliferating cells. Further, in a preferred embodiment, a polynucleotide of the present invention inserted into a growing cell, alone or with or fused to another polynucleotide, is then subjected to an external stimulus (ie, Magnetic, specific small molecules, chemicals, 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 apparently modulated (ie, to increase, decrease, or inhibit the expression of a polynucleotide of the present invention) based on the external stimuli described above.

[0539] The polynucleotides of the present invention may be useful in suppressing the expression of oncogenic genes or antigens. By "suppressing the expression of an oncogenic gene", the suppression of gene transcription and the degradation of gene transcripts (pre-messenger (pre-message) R
NA), intends to inhibit splicing, disrupt messenger RNA, prevent post-translational modification of proteins, disrupt proteins, or inhibit the normal function of proteins.

For topical administration to abnormally growing cells, the polynucleotides of the invention can be administered by any method known to those of skill in the art, including transfection, electroporation, cell Including, but not limited to, microinjection, for example, in a vehicle such as a liposome, lipofectin, or a naked polynucleotide, or any other method described throughout this specification. The polynucleotide of the present invention can be prepared by a known delivery system (for example, a retroviral vector known to those skilled in the art (Gilboa, JV).
irology 44: 845 (1982); Hocke, Nature 32.
0: 275 (1986); Wilson et al., Proc. Natl. Acad. S
ci. USA. 85: 3014); Vaccinia virus system (Chakubarbar)
ty et al., Mol. Cell Biol. 5: 3403 (1985)) or other efficient DNA delivery systems (Yates et al., Nature 313: 812 (198).
5) Not limited to)). These references are exemplary only and are incorporated herein by reference. Deliver specifically to, or transfect, cells that are abnormally growing and leave non-dividing cells (
For spares, it is preferred to utilize a retrovirus or adenovirus (eg, as described in the art or herein) delivery system known to those of skill in the art. Since host DNA replication requires retroviral DNA to integrate,
This retrovirus cannot self-replicate due to the lack of the required retroviral genes for its life cycle. For the polynucleotides of the present invention, such retroviral delivery systems are utilized to target the above genes and constructs to abnormally growing cells and leave non-dividing normal cells.

The polynucleotides of the present invention may be used to control cell proliferative disorders in internal organs, body cavities, etc. by using an imaging device used to guide the injection 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 a disease site at the time of surgical intervention.

“Cell proliferative disorder” means that any human or animal disease or disorder affects any one or any combination of organs, cavities, or body parts. The disease, whether benign or malignant, is characterized by one or more local abnormal growths of cells, or groups of cells, or tissues.

Any amount of a polynucleotide of the invention can be administered, as long as this amount has a biologically inhibitory effect on the growth of the treated cells. Further, more than one polynucleotide of the present invention can be administered simultaneously to the same site. "
"Biologically inhibitory" refers to partial or total growth inhibition as well as a decrease in the rate of proliferation or growth of a cell. A biologically inhibitory dose can be used to assess the effect of a polynucleotide of the invention on tumor growth in a target malignant or abnormally growing cell, animal or animal and cell culture in tissue culture, or It can be determined by any other method known to the person skilled in the art.

The invention further relates to antibody-based therapy, wherein the method comprises administering to a mammalian patient (particularly a human patient) an anti-polypeptide antibody and an anti-polynucleotide antibody to treat one or more of the above disorders. Administering. Methods for producing anti-polypeptide and anti-polynucleotide antibodies (polyclonal and monoclonal) are described in detail elsewhere herein. Such antibodies can be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

An overview of how the antibodies of the present invention can be used therapeutically, for example, see Complement (CDC)
Or binding the polynucleotide or polypeptide of the invention locally or systemically in the body, or by the direct cytotoxicity of antibodies, as mediated by effector cells (ADCC). Some of these approaches are described in more detail below. Given the teachings provided herein, one of skill in the art would understand how to use the antibodies of the present invention without undue experimentation for diagnostic, monitoring or therapeutic purposes.

In particular, the antibodies, fragments and derivatives of the invention treat a subject having or developing a cell proliferative disorder and / or a differentiation disorder as described herein. Useful to Such treatment includes administering a single or multiple doses of the antibody, fragment, derivative, or conjugate thereof.

The antibodies of the present invention may act in combination with other monoclonal or chimeric antibodies, or in a manner that increases the number or activity of effector cells that interact with the lymphokine or hematopoietic growth factor (eg, the antibody). ) Can be used advantageously.

Use of high affinity and / or potent in vivo inhibitory and / or neutralizing antibodies against a polypeptide or polynucleotide, fragment or region thereof of the present invention may be carried out using the polynucleotide or polynucleotide of the present invention. peptide(
(Including fragments thereof) are preferred both for immunoassays for disorders related to and for the treatment thereof. Such an antibody, fragment, or region preferably has an affinity for a polynucleotide or polypeptide, including a fragment thereof. Preferred binding affinities are 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
Includes affinity with a dissociation constant or Kd of less than 5 × 10 ⁻¹³ M, 10 ⁻¹³ M, 5 × 10 ⁻¹⁴ M, 10 ⁻¹⁴ M, 5 × 10 ⁻¹⁵ M, and 10 ⁻¹⁵ M.

Further, the polypeptides of the present invention may be used alone, as fusion proteins, or directly or indirectly in combination with other polypeptides, as described elsewhere herein. Either of the above is useful in inhibiting angiogenesis of proliferating cells or tissues. In a most preferred embodiment, the anti-angiogenic effect described above can be achieved indirectly, for example, through inhibition of hematopoietic tumor-specific cells (eg, tumor-associated macrophages) (Joseph, incorporated by reference,
IB et al., J Natl Cancer Inst, 90 (21): 1648-
53 (1998)). Antibodies to the polypeptides or polynucleotides of the invention can also cause direct or indirect inhibition of angiogenesis (Witte L. et al., Cancer Metastasis, incorporated by reference).
Rev .. 17 (2): 155-61 (1998)).

The polypeptides of the present invention (including protein fusions), or fragments thereof, may be useful in inhibiting proliferating cells or tissues through inducing apoptosis. The polypeptides described above can be used to induce apoptosis of proliferating cells and tissues, either directly or indirectly, such as, for example, death domain receptors (eg, tumor necrosis factor (TNF) receptor-1, CD95 ( Fas / APO
-1), TNF receptor-related apoptosis-mediating protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptors 1 and 2
(See Schulze-Osthoff K, et al., Eur J Biochem 254 (3): 439-59 (1998), which is incorporated herein by reference)). Further, in another preferred embodiment of the present invention, the polypeptide described above may be expressed by other mechanisms (eg, in the activation of other proteins that activate apoptosis) or by expression of the protein alone or by small molecule drugs. Alternatively, apoptosis can be induced through stimulation with either an adjuvant (eg, apoptinin, galectin, thioredoxin, anti-inflammatory proteins) (eg, Mutat, all incorporated herein by reference). Res 400 (1-2): 447-5
5 (1998), Med Hypotheses. 50 (5): 423-33 (
1998), 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 (1998)).

The polypeptides of the invention (protein fusions thereto), or fragments thereof, are useful in inhibiting metastasis of proliferating cells or tissues. Inhibition may be as a direct result of administering the polypeptide or an antibody against the polypeptide, as described elsewhere herein, or indirectly (eg, as is known to inhibit metastasis). Activates expression of a protein (eg, α4 integrin), which can occur (eg, Curr T, incorporated herein by reference).
op Microbiol Immunol 1998; 231: 125-41.
checking). 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 present invention provides a polypeptide (eg, a composition comprising a polypeptide antibody, heterologous nucleic acid, toxin, or prodrug related to a polypeptide or heterologous polypeptide) of the invention, comprising Provided are methods of delivering a polypeptide of the invention to targeted cells expressing the polypeptide. The polypeptides or polypeptide antibodies of the present invention may comprise a heterologous polypeptide, a heterologous nucleic acid, a toxin, or a prodrug, and a hydrophobic,
They may be related through hydrophilic, ionic and / or covalent interactions.

The polypeptides of the invention, protein fusions thereto, or fragments thereof can be directly (eg, propagated when the polypeptides of the invention are “vaccinated”) against the antigens and immunogens described above. Either raise an immune response to respond to sexual antigens and immunogens or indirectly (eg, in activating expression of a protein known to enhance the immune response (eg, a chemokine)) And is useful in enhancing the immunogenicity and / or antigenicity of proliferating cells or tissues.

Cardiovascular Disorders The polynucleotides or polypeptides, or agonists or antagonists, of the invention can be used to treat cardiovascular disorders, including peripheral arterial disease, such as limb ischemia.

[0555] Cardiovascular disorders include arterio-arterial fistulas.
stula), arteriovenous fistula, cerebral arteriovenous congenital anomaly, congenital heart defect (congeni)
tal heart defects), cardiovascular abnormalities such as pulmonary valve atresia, and scimitar syndrome. Congenital heart defects include aortic constriction, triatrial heart, coronary vessel anomalies.
, Cross heart, right thoracic heart, patent ductus arteri
osus), Ebstein malformation, Eisenmenger complex, left ventricular dysgenesis syndrome, left thoracic heart, tetralogy of Fallot, transposition of great arteries, right ventricle of both great vessels, tricuspid regurgitation, remnant ductus arteriosus , And heart septal defect
ts) (eg, aortopulmonary septum defect)
eptal defect, endocardial bed deficiency, Ryutanbache syndrome, trilogy of Fallot, ventricular heart septum (ventricular heart sep)
tal defects))).

Cardiovascular disorders also include arrhythmias, carcinoid heart disease, high cardiac output (hi
gh cardiac output, low cardiac output (low cardiac)
output), heart tamponade (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-infarct cardiac rupture, ventricular septum rupture, heart valve disease, myocardial disease, myocardial ischemia, endocardial effusion, pericarditis (including infarct and tuberculosis), pneumocardiosis, after pericardiotomy Syndrome, right heart disorder, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications (
cardiovascular pregnancy complexity
ns), Scimitar syndrome, cardiovascular syphilis, and cardiovascular tuberculosis (cardiovas)
heart disease such as, for example, C. tuberculosis.

As arrhythmias, sinus arrhythmias, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes syndrome, leg block, sinoatrial block, long QT syndrome, accessory contraction,
Lone-Gangong-Levine Syndrome, Mahemite Premature Excitement Syndrome, Wolf-
Parkinson-White syndrome, sinus dysfunction syndrome, tachycardia, and ventricular fibrillation. Tachycardia includes paroxysmal tachycardia, supraventricular tachycardia, promotion of ventricular specific rhythm, atrioventricular nodal reentrant tachycardia, ectopic atrial tachycardia, ectopic junction tachycardia, sinoatrial node reentrant tachycardia, These include sinus tachycardia, torsades de pointes, and ventricular tachycardia.

[0558] Heart valve diseases include aortic valve dysfunction, aortic stenosis, heart murmur (hea).
murmurs), aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral dysfunction, mitral stenosis, pulmonary valve regurgitation, pulmonary valve dysfunction, pulmonary valve stenosis, tricuspid Examples include atresia, tricuspid dysfunction, and tricuspid stenosis.

The cardiomyopathy includes alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, hypovalvular aortic stenosis, hypovalvular pulmonary stenosis, restricted cardiomyopathy, Chagas cardiomyopathy, intracardiac Membrane fibroelastosis, 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
g) coronary artery disorders.

Cardiovascular disease also includes aneurysms, angiodysplasia, hemangiomatosis, bacterial hemangiomatosis, Hippel-Lindara disease, Kripel-Tornonnay-Weber syndrome, Sturge-Weber syndrome, vasomotor edema, Aortic disorder, Takayasu arteritis, aortic inflammation, Lurish syndrome, arterial obstruction disorder, arteritis, endarteritis (enarter
itis), polyarteritis nodosa, cerebrovascular disease, diabetic vascular disease, diabetic retinopathy, embolism, thrombosis, acrotaxis, hemorrhoids, hepatic vein obstruction disorder, hypertension, hypotension, ischemia, Peripheral vascular disorder, phlebitis, pulmonary vein obstruction disorder, Raynaud's disease, CREST syndrome,
Retinal vein occlusion, scimitar syndrome, superior vena cava syndrome, telangiectasia, telangiectasia ataxia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcers, vasculitis, And vascular disorders such as venous insufficiency.

The aneurysms include dissecting aneurysms, pseudoaneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, cardiac aneurysms, and iliac aneurysms Is mentioned.

Arterial occlusive disorders include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasia, mesenteric vascular occlusion, Moyamoya disease, renal artery occlusion, retinal artery occlusion, and obstructive thrombus Vasculitis.

Cerebrovascular disorders include carotid disorder, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery disorder, cerebral obstruction and thrombosis, Carotid thrombosis, sinus thrombosis, Wallenberg syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage (subaraxhnoid hemorr)
hage), cerebral infarction, cerebral ischemia (including transient), subclavian artery stealing syndrome, periventricular leukomalacia,
Vascular headache, cluster headache, migraine, and vertebrobasi
lar) dysfunction.

[0565] Embolisms include air embolism, amniotic fluid embolism, cholesterol embolism, toe cyanosis syndrome, fat embolism, pulmonary embolism, and thromboembolism. Thrombosis includes coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid thrombosis,
Sinus thrombosis, Wallenberg syndrome, and thrombophlebitis.

[0566] Ischemia includes cerebral ischemia, ischemic colitis, compartment syndrome, precompartment syndrome, myocardial ischemia, reperfusion injury, and peripheral limb ischemia. As vasculitis, aoritis, arteritis, Behcet syndrome, Churg-Strauss syndrome, mucocutaneous lymph node syndrome, obstructive thrombotic vasculitis, irritable vasculitis, Schoenlein-Henoch purpura. ), Allergic cutaneous vasculitis and Wegener's granulomatosis.

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

The polypeptide can be administered using any method known in the art, including direct needle injection at the site of delivery, intravenous injection, topical administration, catheter infusion, biolistic ( biolistic) injections, particle accelerators, gel foam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppository solid pharmaceutical formulations, intraoperative decanting or topical application, aerosol delivery, and the like. However, the present invention is not limited to these. Such methods are known in the art. The polypeptides of the invention can be administered as part of a therapeutic, which can be described in more detail below. The methods of polynucleotide delivery of the present invention are described in more detail herein.

Anti-angiogenic activity The naturally occurring equilibrium between endogenous stimulators and inhibitors of angiogenesis is that in which inhibitory effects predominate. Rastinejad et al., Cell 56.
: 345-355 (1989). In the rare cases where neovascularization occurs under normal physiological conditions (eg, wound healing, organ regeneration, embryonic development, and the female reproductive process), angiogenesis is tightly regulated and spatially and temporally Is determined. Under conditions of pathological angiogenesis (eg, characterizing solid tumor growth), these regulatory controls are not possible. Unregulated angiogenesis becomes pathological and maintains the progression of many neoplastic and non-neoplastic diseases. Many serious diseases are governed by abnormal neovascularization, including solid tumor growth and metastasis, arthritis, some types of ocular disorders, and psoriasis. For example,
Moses et al., Biotech. 9: 630-634 (1991); Folkm.
an et al. Engl. J. Med. 333: 1757-1763 (1995).
); Auerbach et al. Microvasc. Res. 29: 401-4
11 (1985); Folkman, Advances in Cancer.
Academic, edited by Research, Klein and Weinhouse.
Press, New York, pp. 175-203 (1985); Patz,
Am. J. Opthalmol. 94: 715-743 (1982); and F
See the review by Olkman 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 that suggests that solid tumor growth is dependent on angiogenesis. Folkman and Klagsbrunn
, Science 235: 442-447 (1987).

The present invention provides for the treatment of a disease or disorder associated with neovascularization by administration of a polynucleotide and / or polypeptide of the invention, and an agonist or antagonist of the invention. Malignant and metastatic conditions that can be treated with the polynucleotides and polypeptides of the present invention, or agonists or antagonists, include those described herein and other art-known malignancies,
Solid tumors, and cancer (for a review of such disorders, see Fishman et al., M.
edicine, 2nd edition, J.M. B. Lippincott Co. , Phila
delphia (1985)). Accordingly, the present invention provides a method for treating an angiogenesis-related disease and / or disorder comprising administering to a subject in need thereof a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and / or agonist of the present invention. A method of treatment is provided. For example, polynucleotides, polypeptides, antagonists and / or agonists can be utilized in various additional ways to therapeutically treat cancer or tumors. Cancers that can be treated with polynucleotides, polypeptides, antagonists and / or agonists include prostate cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer,
Pancreatic, laryngeal, esophageal, testicular, liver, parotid, bile duct, colon, rectal, cervical, uterine, endometrial, renal, bladder, thyroid cancer Primary tumors and metastases; melanoma; glioblastoma; kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advanced malignancy; and tumors arising from the blood (eg, leukemia) ), But is not limited thereto.
For example, polynucleotides, polypeptides, antagonists and / or agonists can be delivered locally to treat cancers such as skin cancer, head and neck tumor, breast tumor and Kaposi's sarcoma.

In still other aspects, polynucleotides, polypeptides, antagonists and / or agonists can be utilized to treat the surface morphology of bladder cancer, for example, by intravesical administration. Polynucleotides, polypeptides, antagonists and / or agonists can be delivered directly to the tumor or near the site of the tumor via injection or a catheter. Of course, as the skilled artisan will appreciate, the appropriate mode of administration will vary depending on the cancer being treated. Other modes of delivery are discussed herein.

[0572] Polynucleotides, polypeptides, antagonists and / or agonists may be useful in treating other disorders, including angiogenesis, in addition to cancer. These disorders include, but are not limited to: benign tumors (
For example, hemangiomas, acoustic neuromas, neurofibromas, trachoma, and purulent granulomas);
Atherosclerotic plaque; angiogenic diseases of the eye (eg, diabetic retinopathy, immature retinopathy,
Macular degeneration, corneal transplant rejection, neovascular glaucoma, post-lens fibroplasia, rubeosis,
Retinoblastoma, uveitis and pterygium of the eye (Pteryg
ia) (abnormal vascular proliferation)); rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; angiogenesis; granulation; hyperplastic scars (keloids);
Trachoma; vascular adhesion; myocardial angiogenesis; coronary collaterals
arteriovenous malformation; ischemic limb angiogenesis; Osler-Webber syndrome; plaque neovascularization; telangiectasia; hemophilic joint; angiofibromas; Fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.

For example, in one aspect of the present invention, treating hyperplastic scars and keloids comprises administering a polynucleotide, polypeptide, antagonist and / or agonist of the present invention to hyperplastic scars or keloids. A method is provided for doing so.

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 the prophylactic treatment of conditions known to result in the development of hyperplastic scars and keloids (eg, burns), and preferably occurs during the time the proliferative phase progresses (for the first injury). (After about 14 days) but before the onset of hyperplastic scars or keloids. As noted above, the present invention also provides methods for treating ocular neovascularization disorders, including, for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, post lens fibrosis and macular degeneration. I will provide a.

Further, ocular disorders associated with neovascularization that can be treated with the polynucleotides and polypeptides of the present invention (including antagonists and / or agonists) include, but are not limited to, the following: : Angiogenesis glaucoma, diabetic retinopathy, retinoblastoma, retro lens fibroplasia, uveitis, prematurity retinopathy,
Macular degeneration, corneal graft neovascularization, and other inflammatory diseases of the eye, tumors of the eye, and diseases associated with neovascularization of the choroid or iris. For example, Waltman
Et al., Am. J. Ophthal. 85: 704-710 (1978) and Ga
rtner et al., Surv. Ophthal. 22: 291-212 (1978)
See the review by.

Thus, in one aspect of the present invention, corneal neovascularization (including the step of 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 corneal transplant neovascularization). Briefly, the cornea is tissue that usually lacks blood vessels. However, in certain pathological conditions, capillaries can extend from the marginal pericorneal plexus to the cornea. When the cornea becomes vascularized, the cornea also becomes cloudy, causing a decline in the patient's vision. The cornea becomes completely opaque (o
In the event of pacitate, vision loss can be complete. A wide variety of obstacles
For example, it can result in 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 (of any cause), toxicity and malnutrition, and as complications of wearing contact lenses.

In a particularly preferred embodiment, the invention is prepared for topical administration in saline (in combination with any preservatives and antibacterials commonly used in ophthalmic preparations) and It can be administered in a dosage form. Solutions or suspensions are prepared in pure form and may be administered several times a day. Alternatively, the 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, an anti-angiogenic factor or an anti-angiogenic composition may be utilized as an adjunct to conventional steroid therapy. Topical treatment may also be useful prophylactically in corneal lesions known to have a high potential to induce an angiogenic response (eg, chemical burns). In these cases, treatment (possibly combined with steroids) can be started immediately to help prevent subsequent complications.

In one embodiment, the compounds described above can be injected by an ophthalmologist directly under the guidance of a microscope into the corneal stroma. While the preferred injection site may vary in the form of individual lesions, 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). ). In most cases, this will be peri-perimeter (peri) to "protect" the cornea from advancing blood vessels.
limbic) including corneal injections. This method can also be used immediately after corneal injury to prevent corneal neovascularization prophylactically. In this situation, the substance
It can be injected between the corneal lesion and its unwanted potential limbal blood supply into the perilimbic cornea. Such methods can also be used to prevent capillary infiltration of the implanted cornea in a similar manner. In a sustained release form, injections may be 2 to 3
Only three times may be required. Steroids may also be added to the injection solution to reduce inflammation resulting from the injection itself.

In another aspect of the invention, angiogenesis comprising administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and / or agonist to inhibit the formation of blood vessels. Methods are provided for treating glaucoma. In one embodiment, the compound may be administered topically to the eye to treat early forms 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 may also be located at any location so that the compound is continuously released into the aqueous humor. In another aspect of the invention, the method comprises administering to the patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and / or agonist to the eye such that blood vessel formation is inhibited.
Methods are provided for treating proliferative diabetic retinopathy.

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

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

Further, disorders that can be treated with the polynucleotide, polypeptide, agonist and / or agonist include, but are not limited to: hemangiomas, arthritis, psoriasis, hemangiofibromas, atherosclerotic plaque , Delayed wound healing, granulation, hemophilic joints, hyperplastic scars, pseudoarticular fractures, Osler-Weber syndrome, purulent granulomas, scleroderma, trachoma,
And vascular adhesion.

Further, disorders and / or conditions that can be treated with the polynucleotides, polypeptides, agonists and / or agonists include, but are not limited to: solid tumors, blood born Tumors (eg, leukemia), tumor metastases, Kaposi's sarcoma, benign tumors (eg, hemangiomas, acoustic neuromas, neurofibromas, trachoma, and purulent granulomas), rheumatoid arthritis,
Psoriasis, angiogenic diseases of the eye (eg, diabetic retinopathy, retinopathy of prematurity, macular degeneration,
Corneal transplant rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uveitis), delayed wound healing, endometriosis,
Angiogenesis, granulation, hyperplastic scars (keloids), pseudoarticular fractures, scleroderma, trachoma, vascular adhesions, neovascularization of myocardium, coronary colla
terals), collateral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber syndrome, plaque neovascularization, telangiectasia, hemophilic joints, hemangiofibromas, Fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control drugs (by controlling menstruation, by preventing angiogenesis required for embryo implantation), pathogenic consequences ( For example, cat scratch disease (Rochel minalia quintosa), ulcer (H
Diseases with angiogenesis (e.g., E. pylori), bartonellosis and bacterial hemangioma symptoms).

In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after sexual intercourse and fertilization has occurred, and thus is an effective method of birth control, presumably “post-hoc Provide a method. Polynucleotides, polypeptides, agonists and / or agonists may also be used in controlling menstruation or may be administered either as a peritoneal lavage in the treatment of endometriosis or for peritoneal transplantation.

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

The polynucleotide, polypeptide, agonist and / or agonist may be
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 spread of disease to surrounding tissue. To remove or remove the tumor, it can be utilized to coat or spray the area. In one aspect of the invention, the composition (eg, in the form of a spray) can be delivered via an endoscopic procedure to coat a tumor or to inhibit angiogenesis at a desired location. . In yet another aspect of the present invention, a surgical mesh coated with the anti-angiogenic composition of the present invention can be utilized in any procedure where a surgical mesh can be utilized. For example, in one embodiment of the present invention, a surgical mesh with an anti-angiogenic composition may be used to provide support for the structure and to release a certain amount of anti-angiogenic factors. It can be utilized during resection surgery (eg, after colectomy).

In a further aspect of the invention, polynucleotides, polypeptides, agonists and / or agonists at the resected margin of the tumor after resection so that local recurrence of the cancer and formation of new blood vessels at the site are inhibited. A method is provided for treating a tumor resection site, comprising the step of administering. In one embodiment of the invention, the anti-angiogenic compound is administered directly to the site of tumor excision (eg, smearing, brushing, or otherwise excising the tumor margin with an anti-angiogenic compound). Applied by coating). Alternatively, the anti-angiogenic compound is
It can be incorporated into known surgical pastes before administration. In a particularly preferred embodiment of the invention, the anti-angiogenic compound is applied after hepatectomy for malignancy and after neurosurgery.

In one aspect of the invention, the polynucleotide, polypeptide, agonist and / or agonist is administered to the resection margin of a wide variety of tumors, including, for example, breast, colon, brain and liver tumors. Can be done. For example, in one embodiment of the present invention, the anti-angiogenic compound, after resection, at the site of a neurological tumor,
The administration can be such that the formation of new blood vessels at the site is inhibited.

The polynucleotides, polypeptides, agonists and / or agonists of the invention can also be administered with other angiogenic factors. Representative examples of other anti-angiogenic factors include: anti-invasive factors, retinoic acid and its derivatives, paclitaxel, suramin, tissue inhibitors of metalloproteinase-1,
Tissue inhibitor of metalloproteinase-2, plasminogen activator
1. Plasminogen activator-2 and various forms of lighter "group d" transition metals.

[0590] Lighter "d group" transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium and tantalum species. Such transition metal species can form a transition metal complex. Suitable complexes of the aforementioned transition metal species include oxo transition metal complexes.

[0591] Representative examples of vanadium complexes include oxovanadium complexes such as vanadate complexes and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate (including vanadyl sulfate hydrates such as vanadyl sulfate monohydrate and vanadyl sulfate trihydrate).

[0592] Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxotungsten 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)
Oxides. Suitable oxomolybdenum complexes include molybdate,
Molybdenum oxide and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide,
Molybdenum (VI) oxide and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include, for example, hydroxo derivatives 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;
ated) chitin derivatives (prepared from the queen crab shell) (Murata et al., Cancer Res. 51: 22-26,199).
1); sulfated polysaccharide peptidoglycan complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen and tamoxifen citrate); staurosporine; modulators of matrix metabolism (eg, Proline analog, cis hydroxyproline, d, L-3,4
-Dehydroproline, thiaproline, α, α-dipyridyl, aminopropionitrile fumarate); 4-propyl-5- (4-pyridinyl) -2 (
3H) -oxazolone; methotrexate; mitozantrone; heparin; interferon; 2 macroglobulin-serum; ChIMP-3 (Pavloff et al.
J. Bio. Chem. 267: 17321-17326, 1992); chymostatin (Tomkinson et al., Biochem J. 286: 475-480).
, 1992); cyclodextrin tetradecasulfate; eponemycin (
Eponmycin); Camptothecin; Fumagillin (Ingber et al., Nature 348: 555-557, 1990).
Gold sodium thiomalate ("GST"; Matsubara and Ziff);
, J. et al. Clin. Invest. 79: 1440-1446, 1987); anti-collagenase-serum; α2-antiplasmin (Holmes et al., J. Biol. C).
hem. 262 (4): 1659-1664, 1987); bisantrene (Na
lonaliter Institute; lobenzarit disodium (N- (2) -carboxyphenyl-4-chloroanthronic acid (chloro)
roanthronic acid) disodium or "CCA"; Ta
Keuchi et al., Agents Actions 36: 312-316,19.
92); Thalidomide; Angostatic steroid (Angostatic)
AGM-1470; carboxinaminolumidazole (c
and a metalloproteinase inhibitor (eg, BB94).

Diseases at the Cell Level Diseases associated with increased cell survival or inhibition of apoptosis that can be treated or detected by the polynucleotides or polypeptides and / or antagonists or agonists of the invention include cancer (eg, follicles) Lymphoma, p53
Carcinomas with mutations, and hormone-dependent tumors, which are: colon cancer, cardiac tumor, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, gastric cancer, Including, but not limited to, neuroblastoma, myxoma, fibroid, lymphoma, endothelioma, osteoblastoma, giant cell tumor, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer Autoimmune disorders (eg, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease (Behcet's disease)
's disease), Crohn's disease, polymyositis, systemic lupus erythematosus and immune-associated glomerulonephritis and rheumatoid arthritis) and viral infections (eg, herpes virus, poxvirus and adenovirus), inflammation, graft-versus-host disease , Acute graft rejection, as well as chronic graft rejection. In a preferred embodiment, the polynucleotides or polypeptides and / or agonists or antagonists of the invention are used to inhibit the growth, progression and / or metastasis of cancer, especially those listed above. .

Additional diseases or conditions associated with increased cell survival that can be treated or detected by the polynucleotides or polypeptides of the invention, or agonists or antagonists, include the progression and / or metastasis of malignant diseases and the associations as follows But not limited to: leukemia (acute leukemia (eg, acute lymphocytic leukemia, acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia) And chronic leukemia (eg, chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (eg, Hodgkin's disease and non-Hodgkin's disease), multiple bone marrow Tumors, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors (
Sarcomas and carcinomas (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, hemangiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphatic endothelioma, periosteoma, mesothelioma , Ewing tumor, leiomyosarcoma,
Rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, cystic adenocarcinoma, medulla Like carcinoma, primary bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, fetal carcinoma,
Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pine Paleomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas
), Melanomas, neuroblastomas, and retinoblastomas).

Diseases associated with increased apoptosis that can be treated or detected by the polynucleotides or polypeptides of the invention and / or agonists or antagonists include: AIDS; neurodegenerative disorders (eg, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumors or previously related diseases); autoimmune disorders (eg, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease (B
ehcet's disease), Crohn's disease, polymyositis, systemic lupus erythematosus and immune-associated glomerulonephritis and rheumatoid arthritis), myelodysplastic syndrome (eg, aplastic anemia), graft versus host disease, ischemic Injury (such as caused by myocardial infarction, stroke and reperfusion injury), liver injury (eg, hepatitis-related liver injury, ischemia / reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxicants 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, a polynucleotide or polypeptide, and / or agonist or antagonist of the present invention is used for therapeutic purposes, for example, for wound healing purposes. Processes are provided for utilization to stimulate epithelial cell proliferation and basal keratinocytes, and to stimulate hair follicle production and skin wound healing. Polynucleotides or polypeptides of the present invention, as well as agonists or antagonists, may be clinically useful in stimulating wound healing, including: surgical wounds, resected wounds, deep wounds (damaging the dermis and epidermis) Ophthalmic tissue wounds, dental tissue wounds, oral wounds, diabetic ulcers, skin ulcers, elbow ulcers, arterial ulcers, venous stasis ulcers, exposure to heat or chemical burns, And other abnormal wound healing conditions (eg, uremia, malnutrition, vitamin deficiency, and complications associated with steroids, radiotherapy and systemic treatment with antineoplastic drugs and antimetabolites. Nucleotides or polypeptides, and / or agonists or antagonists, are used to promote skin healing following skin loss. Get.

The polynucleotides or polypeptides and / or agonists or antagonists of the invention may be used to increase the attachment of skin grafts to the wound bed and to stimulate re-epithelialization from the wound bed Can be used for The following is a non-exhaustive list of grafts in which the polynucleotides or polypeptides, agonists or antagonists of the invention can be used to increase adhesion to the wound bed: autograft, artificial skin, allograft Piece (allograft)
Autograft, autoepdermic graft, avascular graft, Blair-Brown graft, bone graft, embryonic tissue graft, dermal graft, delayed graft, skin graft, Epidermal graft, fascial graft,
Full thickness skin graft, xenograft, xenograft, homologous graft
t), proliferative grafts, lamellar grafts, reticular grafts, mucosal grafts, Orie-Tielsch grafts, omenpal grafts, patch grafts, stem grafts, all Penetrating graft, stratified skin graft, stratified skin graft. The polynucleotides or polypeptides and / or agonists or antagonists of the present invention can be used to promote skin strength and improve the appearance of aged skin.

The polynucleotides or polypeptides and / or agonists or antagonists of the present invention also cause changes in hepatocyte proliferation and epithelial cell proliferation in lung, breast, pancreas, stomach, small intesting, and large intestine it is conceivable that. The polynucleotides or polypeptides, and / or agonists or antagonists of the present invention may be administered to epithelial cells (eg, sebocytes, hair follicles, liver parenchymal cells, alveolar epithelial cells type II (typ)
e II pneumocyte), mucin-producing goblet cells, and other epithelial cells,
And promote the growth of skin, lungs, liver, and their ancestors contained within the gastrointestinal tract). A polynucleotide or polypeptide and / or agonist or antagonist of the present invention can promote the growth 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, chemotherapeutic treatment or viral infection. The polynucleotides or polypeptides and / or agonists or antagonists of the invention may have cytoprotective effects on the small intestinal mucosa. The polynucleotides or polypeptides and / or agonists or antagonists of the invention may also be used in mucositis (oral ulcers) resulting from chemotherapy and viral infections.
Can stimulate healing of

The polynucleotides or polypeptides and / or agonists or antagonists of the present invention may be used to fully regenerate the skin (ie, hair follicles, sweat glands, and skin) in complete and partial thickness skin defects, including burns. Regrowth of sebaceous glands),
It can further be used in the treatment of other skin defects such as psoriasis. The polynucleotides or polypeptides and / or agonists or antagonists of the present invention may be used in epidermolysis bullosa (the epidermis to the underlying dermis, which produces frequent open and painful blisters by promoting re-epithelialization of these lesions) Deficiency in adhesion). The polynucleotides or polypeptides and / or agonists or antagonists of the present invention also treat gastric and duodenal ulcers and are healed by scarring of the lining of the mucosa and more rapidly regenerating the lining of the glandular and duodenal mucosa. Can be used to help.
Inflammatory bowel diseases (eg, Coulomb's disease and ulcerative colitis) are diseases that result in disruption of the mucosal surface of the small intestine or colon, respectively. Thus, the polynucleotides or polypeptides, and / or agonists or antagonists of the present invention may promote resurfacing of mucosal surfaces to aid more rapid healing and prevent the progression of inflammatory bowel disease Can be used to Treatment with the polynucleotides or polypeptides of the invention and / or agonists or antagonists is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract, and the intestinal mucosa may be ingested or harmful. It can be used to protect against substances or after surgery. The polynucleotide or polypeptide, and / or agonist or antagonist of the present invention can be used to treat a disease associated with the expression of the polynucleotide of the present invention.

In addition, 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 to prevent or treat acute or chronic lung injury, and can inhibit alveoli and bronchioles. (Brochiola) Can promote epithelial repair. For example, emphysema that results in a progressive loss of aveoli and inhalation insults that result in necrosis of bronchiolar epithelium and alveoli (ie, resulting from smoke inhalation and burns) are subject to polynucleotides or polynucleotides of the invention. It can be effectively treated using peptides, and / or agonists or antagonists. 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 cells type II, which can be used to stimulate vitreous membrane disease in premature infants (eg, , 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 hepatic parenchymal cells, and are therefore capable of stimulating liver disease and conditions, such as fulminant hepatic failure caused by cirrhosis, It can be used to alleviate or treat hepatitis virus and toxic agents (ie, liver damage caused by acetaminophen, carbon tetrachloride, and other hepatotoxins known in the art).

Further, the polynucleotides or polypeptides, and / or agonists or antagonists of the 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 functions remain, the polynucleotide or polypeptide, and / or agonist or antagonist of the present invention may It can be used to maintain its islet function, such as to alleviate, delay or prevent persistent expression. Also, the polynucleotides or polypeptides and / or agonists or antagonists of the present invention can be used as an aid in islet cell transplantation to improve or promote islet cell function.

Neurological Diseases Neurological diseases that can be treated with the compositions (eg, polypeptides, polynucleotides and / or agonists or antagonists) of the present invention include, but are not limited to, the following: Systemic injuries and diseases or injuries that result in either axonal severing, neuronal loss or degeneration or demyelination. Nervous system lesions that can be treated in patients (including human and non-human mammal patients) in accordance with the present invention include lesions of any of the following central (including spinal cord, brain) or peripheral nervous systems: (1) An ischemic lesion in which lack of oxygen in parts of the nervous system results in nervous system injury 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 (eg, lesions that cut parts of the nervous system or compressive injuries); (3) parts of the nervous system are nervous system-related malignancies Or a malignant lesion that has been destroyed or injured by malignant tissue that is either malignant tumor from non-nervous system tissue; or (4) a portion of the nervous system Are injured or infected by human immunodeficiency virus, shingles or herpes simplex virus, or infectious foci associated with Lyme disease, tuberculosis, syphilis; (5) part of the nervous system is Parkinson's disease, Alzheimer's disease Degenerative lesions that are destroyed or injured as a result of degenerative processes, including but not limited to degeneration associated with the disease, Huntington's chorea or amyotrophic lateral sclerosis (ALS); Nutritional disorders or metabolism, including but not limited to vitamin B12 deficiency, folate deficiency, Wernicke's disease, tobacco-alcoholic amblyopia, Marchiafava-Vinami disease (early degeneration of the corpus callosum) and alcoholic cerebellar degeneration Lesions associated with a nutritional disease or disorder that are destroyed or injured by the disorder; Neurologic lesions associated with systemic diseases including, but not limited to, systemic lupus erythematosus, cancer or sarcoidosis; (8) caused by toxic substances including alcohol, lead or certain neurotoxins And (9) multiple sclerosis, human immunodeficiency virus-associated myelopathy, transversal myelopathy
opathy) or demyelinating lesions that are destroyed or injured by demyelinating diseases including, but not limited to, pathogenesis, progressive multifocal leukoencephalopathy, and central pontine myelin lysis.

In a preferred embodiment, the polypeptides, polynucleotides or agonists or antagonists of the invention are used to protect neurons from the deleterious effects of cerebral hypoxia. According to this embodiment, the compositions of the invention are used to treat or prevent neuronal damage associated with cerebral hypoxia. In one aspect of this example, a polypeptide, polynucleotide or agonist or antagonist of the invention is used to treat or prevent neuronal damage associated with cerebral ischemia. In another aspect of this embodiment, the polypeptide of the present invention,
The polynucleotide or agonist or antagonist is used to treat or prevent neuronal damage associated with cerebral infarction formation. In another aspect of this embodiment, a polypeptide, polynucleotide or agonist or antagonist of the invention is used to treat or prevent stroke-related neuronal damage. In yet another aspect of this embodiment, a polypeptide, polynucleotide or agonist or antagonist of the invention is used to treat or prevent neuronal damage associated with a heart attack.

The compositions of the present invention that are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and without 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 culture; (3) increased sprouting of neurons in culture or in vivo; (3) increased production of neuron-associated molecules (eg, choline acetyltransferase or acetylcholinesterase for motor neurons) in culture or in vivo;
2.) Reduced symptoms of neuronal dysfunction in vivo. Such an effect can be measured by any method known in the art. In a preferred and non-limiting embodiment, the increased survival of neurons is determined, for example, by methods described herein or otherwise known in the art (eg, Arakawa et al. (J. Nurt).
osci. 10: 3507-3515 (1990)); increased sprouting of neurons can be measured by methods known in the art (eg, Pestronk et al. (Exp. Neurol. 70: 65-82 (
1980)) or Brown et al. (Ann. Rev. Neurosci. 4: 1).
7-42 (1981)); increased production of neuron-associated molecules can be detected using bioassays using techniques known in the art and depending on the molecule being measured. Motor neuron dysfunction can be measured by an enzymatic assay, antibody binding, Northern blot assay, etc .; Can be measured by

In certain embodiments, motor neuron disorders that can be treated according to the present invention include disorders such as infarction, infection, exposure to toxicants, trauma, surgical damage, degenerative or malignant diseases, motor neurons and Diseases that selectively affect other compositions of the nervous system include, but are not limited to, amyotrophic lateral sclerosis, and include, but are not limited to: progressive Spinal muscular atrophy,
Progressive bulbar palsy, primary lateral sclerosis, pediatric and juvenile muscular atrophy, childhood progressive bulbar palsy (Fazio-Londe syndrome), gray and white poliomyelitis syndrome (post polio syndrome) and Hereditary Motor Sensory Neuropathy
thy) (Charcot-Marie-Tooth disease).

(Infectious Disease) The polynucleotide or polypeptide, and / or agonist or antagonist of the present invention can be used to treat or detect an infectious agent. For example, an infectious disease can be treated by increasing the immune response, particularly by increasing the proliferation and differentiation of B and / or T cells. The immune response can be raised either by raising an existing immune response or by starting a new immune response. Alternatively, a polynucleotide or polypeptide of the invention and / or agonist or antagonist may also directly inhibit infectious agents, without necessarily eliciting an immune response.

Viruses are an example of an infectious agent that can cause a disease or condition that can be treated or detected by a polynucleotide or polypeptide of the present invention and / or agonist or antagonist. Examples of viruses include the following D
Examples of viruses include, but are not limited to, viruses and virions of NA and RNA: arbovirus, adenoviridae, arenaviridae, arterivirus, birnaviridae, bunyaviridae, Calciviridae, Sircoviridae (Circoviridae),
Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (eg, cytomegalovirus, herpes simplex, herpes zoster), Mononegavirus (Mononegavirus) (
For example, paramyxoviridae, measles virus, rhabdoviridae), orthomyxoviridae (eg, influenza A, influenza B, and parainfluenza), papillomavirus, Papovaviridae, parvoviridae, picornavirus. Family, Poxviridae (
Eg, smallpox or vaccinia), reoviridae (eg, rotavirus),
Retroviridae (HTLV-I, HTLV-II, lentivirus), and Togaviridae (e.g., rubivirus). Viruses that fall into 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, cornea) Inflammation), chronic fatigue syndrome, hepatitis (types A, B, C, E, chronic activity, delta), Japanese encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections ( AIDS), pneumonia, Burkitt's lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, cold, polio, leukemia, rubella, sexually transmitted disease, skin disease (eg, capoge, wart)
, And viremia. Any of these conditions or diseases can be treated or detected using the polypeptides or polynucleotides of the present invention, or agonists or antagonists. In certain embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat: meningitis, dengue, 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 unresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment, the polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat AIDS.

Similarly, bacterial or fungal factors that can cause a disease or condition and that can be treated or detected by the polynucleotides or polypeptides and / or agonists or antagonists of the present invention are the following Gram-negative and Gram- Positive bacteria and include but are not limited to Bacteriaceae and fungi: Actinomycetales (eg, Corynebacterium)
, Mycobacterium, Norcardia), Cryptococc
us neoformans, Aspergillosis, Bacillac
eae (eg, Anthrax, Clostridium), Bactero
idaceae, Blastomycosis, Bordetella, Bor
relia (eg, Borrelia burgdorferi, Bruce
llosis, Candidiasis, Campylobacter, Coc
Cidioidomycosis, Cryptococcosis, Derma
tocycoses, E.C. coli (eg, enterotoxin-producing (Ent)
erotoxigenic) E. coli, and enterohemorrhagic E. coli. coli), E
nanobacteriaaceae (Klebsiella, Salmone
lla (eg, Salmonella typhi, and Salmonell
la paratyphi), Serratia, Yersinia), Ery
sipelothrix, Helicobacter, Legionellos
is, Leptospirosis, Listeria, Mycoplasma
tales, Mycobacterium leprae, Vibrioch
oleae, Neisseriaceae (for example, Acinetobacter)
er, Gonorrhea, Menigococcal), Meisseria
meningitidis, Pasteurellacea infections (eg, Actinobacillus, Heamophilus (eg, Heamophilus)
opilis influenza B), Pasteurella), Pseud
omonas, Rickettsiaceae, Chlamydiaceae,
Syphilis, Shigella spp. , Staphylococca
1, Meningiococcal, Pneumococcal and Street
ptococcal (eg Streptococcus pneumoni)
ae and group B Streptococcus). These bacterial or fungal families can cause the following diseases or conditions, including but not limited to:
Bacteremia, endocarditis, ocular infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (eg, AIDS-related infections), peryungitis, prosthesis-related infections, Reiter's disease Respiratory tract infections (eg, pertussis or pyometra), sepsis, Lyme disease, cat scratching disease, dysentery, paratyphoid fever, food poisoning, typhoid fever, pneumonia, gonorrhea, meningitis (eg, meningitis types A and B), Chlamydia, syphilis, diphtheria, leprosy, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, rheumatic fever, scarlet fever, sexually transmitted diseases, skin diseases (eg, cellulitis, dermatomycosis (dermatoc)
ycoses)), toxicemia, urinary tract infections, wound infections. Any of these conditions or diseases may be treated or detected using the polypeptides or polynucleotides, agonists or antagonists of the invention. In certain embodiments, the polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat: tetanus, diphtheria, botulism and / or meningitis type B.

In addition, parasites that cause a disease or condition that can be treated or detected by a polynucleotide or polypeptide and / or agonist or antagonist of the present invention include, but are not limited to, the following families or classes: : Amoebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dinucleomoebias, Diplomacy, Ectoparasitic, Giardia flagellosis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis , Trypanosomiasis, and Trichomonas and sporeworms (eg, Plasmodium vivax, Plasmodium falciparum, Plasmodium vivax and Oval malaria parasite). These parasites can cause a variety of diseases or conditions, including but not limited to: scabies, tsutsugamushi disease, ocular infections, bowel disease (eg, dysentery, giardiasis), liver disease, lung Diseases, opportunistic infections (eg, AIDS-related), malaria,
Pregnancy complications, and toxoplasmosis. Any of these conditions or diseases can be treated or detected using the polypeptides or polynucleotides of the present invention, or agonists or antagonists. In certain embodiments, a polynucleotide, polypeptide, or agonist or antagonist of the invention is used to treat malaria.

Preferably, treatment with a polypeptide or polynucleotide of the invention, and / or agonist or antagonist, comprises administering to the patient an effective amount of the polypeptide or removing cells from the patient to obtain a polypeptide of the invention. Nucleotides may be supplied to the cells and the engineered cells returned to the patient (ex vivo treatment). Further, the polypeptides or polynucleotides of the present invention can be used as an antigen in a vaccine to elicit an immune response against an infectious disease.

(Regeneration) Cells can be differentiated, expanded and attracted using the polynucleotides or polypeptides and / or agonists or antagonists of the invention to direct tissue regeneration (Science 276: 59- 87 (1997)). Using tissue regeneration, birth defects, trauma (wounds, burns, incisions, or ulcers), aging, diseases (eg, osteoporosis, osteoarthritis)
thritis, periodontal disease, liver failure), surgery, including cosmetic surgery, fibrosis, reperfusion injury, or tissue damaged by systemic cytokine damage.

Tissues that can be regenerated using the present invention include: Organs (eg,
Pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal, or cardiac),
Vasculature (including vessels and lymph), nerves, hematopoiesis, and skeleton (bones, cartilage, tendons,
And ligaments) tissue. Preferably, regeneration occurs without scarring or with reduced scarring. Regeneration may also include angiogenesis.

In addition, the polynucleotides or polypeptides, and / or agonists or antagonists of the present invention may increase the regeneration of difficult-to-heal tissues. For example, increasing tendon / ligament regeneration speeds recovery time after injury. A polynucleotide or polypeptide, and / or agonist or antagonist of the present invention may also be used prophylactically in an attempt to avoid injury. Specific diseases that can be treated include tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of non-healing wound tissue regeneration is the compression ulcer (pr
ulcers, as well as ulcers associated with vascular insufficiency, surgical wounds, and traumatic wounds.

Similarly, nerve and brain tissue can also be regenerated by using the polynucleotides or polypeptides of the invention and / or agonists or antagonists to proliferate and differentiate nerve cells. Diseases that can be treated using the present methods include central and peripheral nervous system disorders, neurological disorders, or mechanical and traumatic disorders such as spinal cord disorders, head trauma, cerebrovascular disease, and stroke (
stokes)). In particular, 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's) Disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome) can all be treated with a polynucleotide or polypeptide of the invention and / or an agonist or antagonist.

(Chemotaxis) The polynucleotide or polypeptide and / or agonist or antagonist of the present invention may have chemotactic activity. Chemotactic molecules convert cells (eg, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells, and / or endothelial cells) into specific sites in the body (eg, At sites of inflammation, infection, or hyperproliferation). The recruited cells may then repel and / or heal certain traumas or abnormalities.

The polynucleotides or polypeptides and / or agonists or antagonists of the invention may increase the chemotactic activity of a particular cell. These chemotactic molecules are then used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to specific locations in the body. obtain. For example, chemotactic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. The chemotactic molecule of the present invention can also attract fibroblasts, which can be used to treat wounds.

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

(Binding Activity) The polypeptide of the present invention can be used for screening for a molecule that binds to the polypeptide or a molecule to which the polypeptide binds. Binding of a polypeptide to a 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 a low molecule is mentioned.

Preferably, the molecule is closely related to the natural ligand of the polypeptide (eg, a fragment of the ligand) or to a natural substrate, ligand, structural or functional mimic (Coligan et al., Current). Protocol
s in Immunology 1 (2): see Chapter 5 (1991)). Similarly, a molecule can be a natural receptor to which a polypeptide binds, or at least a fragment of a receptor that can be bound by a polypeptide (eg,
Active site). In each 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, Drosop
hira, or E. coli-derived cells. The cell expressing the polypeptide (or the cell membrane containing the expressed polypeptide) can then preferably contain the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule. Contact with a potential test compound.

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

Alternatively, assays can be performed using cell-free preparations, polypeptides / molecules attached to a solid support, chemical libraries, or natural product mixtures. The assay also comprises mixing a solution comprising the polypeptide with the candidate compound,
It may simply involve measuring the activity or binding of the polypeptide / molecule, and comparing the activity or binding of the polypeptide / molecule to a standard.

Preferably, an ELISA assay may use a monoclonal or polyclonal antibody to measure the level or activity of a polypeptide in a sample (eg, a biological sample). Antibodies can be produced by either direct or indirect binding to a polypeptide, or by competition for a substrate with the polypeptide.
The level or activity of the polypeptide can be measured.

In addition, receptors to which the polypeptides of the present invention bind can be identified using any number of methods known to those of skill in the art, including ligand panning and FACS sorting (Coligan et al., C
current Protocols in Immun. , 1 (2), Chapter 5,
(1991)). For example, expression cloning is used,
There, the polyadenylated RNA is converted into cells responsive to the polypeptide (
For example, 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 OS cells or other cells that are not responsive to the polypeptide. Transfected cells grown on glass slides are exposed to a polypeptide of the invention after being labeled. The polypeptide may be labeled by various means, including iodination, or inclusion 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, ultimately resulting in a single clone encoding the putative receptor.

As an alternative approach for receptor identification, labeled polypeptides can be photoaffinity linked to cell membranes or extract preparations that express the receptor molecule. The crosslinked material is degraded by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptor for the polypeptide can be excised, broken down into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing was used to design a set of degenerate oligonucleotide probes, c
DNA libraries can be screened to identify the gene encoding the putative receptor.

In addition, the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as “DNA shuffling”) can be utilized to modulate the activity of a polypeptide of the present invention. , Thereby effectively producing agonists and antagonists of the polypeptides of the invention. In general, U.S. Pat.
Nos. 811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P.A. A. Curr
. Opinion Biotechnol. 8: 724-33 (1997); H
arayama, S .; Trends Biotechnol. 16 (2): 76
-82 (1998); Hansson, L .; O. J. et al. Mol. Biol. 2
87: 265-76 (1999); and Lorenzo, M .; M. And B
lasco, R.A. Biotechniques 24 (2): 308-13 (
1998), each of which is hereby incorporated by reference. In one embodiment, modification of the polynucleotides and corresponding polypeptides of the invention can be achieved by DNA shuffling.
DNA shuffling involves the assembly of two or more DNA segments into the desired polynucleotide sequence of a molecule of the present invention by homologous recombination or site-specific recombination. In another embodiment, the polynucleotides and corresponding polypeptides of the present invention are prepared using an error-prone PCR,
It can be modified by subjecting it to random mutagenesis by random nucleotide insertion or other methods. In another embodiment, one or more components, motifs, sections, domains, domains, fragments, etc., of the polypeptide of the invention are one or more components, motifs, partitions (one or more) of one or more heterologous molecules. s
section, domain, fragment, and the like. In a preferred embodiment, the heterologous molecule is a family member. In a further preferred embodiment, the heterologous molecule is, for example, a platelet-derived growth factor (PD
GF), insulin-like growth factor (IGF-I), transforming growth factor (
TGF) -α, epidermal growth factor (EGF), fibroblast growth factor (FGF), TG
F-β, bone morphogenic protein (BMP)
-2, BMP-4, BMP-5, BMP-6, BMP-7. Activin (act
ivin) A and B, decapentaplegi
c) (dpp), 60A, OP-2, dorsalin, growth differentiation factor (GDF), nodule, MIS, inhibin-α, TGF-β
1, growth factors such as TGF-β2, TGF-β3, TGF-β5, and glial-derived neurotrophic factor (GDNF).

[0631] 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.

The present invention further provides methods of screening compounds to identify those that modulate the action of a polypeptide of the present invention. An example of such an assay involves combining a mammalian fibroblast, a polypeptide of the invention, the compound to be screened and 3 [H] thymidine under cell culture conditions under which fibroblasts normally grow. . Control assays can be performed in the absence of the compound to be screened, and the uptake of 3 [H] thymidine in each case compared to the amount of fibroblast proliferation in the presence of the compound. The determination may determine whether the compound stimulates proliferation. The amount of fibroblast proliferation
It can be measured by liquid scintillation chromatography which measures incorporation of 3 [H] thymidine. Both agonist and antagonist compounds can be identified by this procedure.

In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the invention is incubated with a labeled polypeptide of the invention in the presence of the compound. The ability of the compound to enhance or block this interaction can then be measured. Alternatively, the response of the known second messenger system and the receptor following the interaction of the compound to be screened is measured, and the ability of the compound to bind 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 can be used to treat or treat diseases by activating or inhibiting polypeptides / molecules or to produce specific results in patients (eg, vascular growth). . In addition, assays may find factors that can inhibit or enhance the production of a polypeptide of the present invention from suitably engineered cells or tissues. Accordingly, the invention includes a method of identifying a compound that binds to a polypeptide of the invention, comprising the steps of: (a)
Incubating the candidate binding compound with the polypeptide of the invention; and (b) determining whether binding has occurred. In addition, the invention includes a method of identifying an agonist / antagonist comprising the steps of: (a) incubating a candidate compound with a polypeptide of the invention; and (b) assaying for biological activity. And (b) determining whether the biological activity of the polypeptide has been altered.

Also, by using the β-pleated sheet region included in the polypeptide sequence of the protein, a molecule that binds to the polypeptide of the present invention can be experimentally identified. Accordingly, certain embodiments of the present invention include the amino acid sequence of each β-pleated sheet region in the disclosed polypeptide sequences, or the amino acid sequence of each β-pleated sheet region in the disclosed polypeptide sequences. Consisting of a polynucleotide encoding a polypeptide. Further embodiments of the invention encode a polypeptide comprising any combination or all contained in the polypeptide sequence of the invention, or consisting of any combination or all contained in the polypeptide sequence of the invention. To a polynucleotide. A further preferred embodiment of the invention comprises the amino acid sequence of each β-pleated sheet region in one of the polypeptide sequences of the invention or the amino acid sequence of each β-pleated sheet region in one of the polypeptide sequences of the invention For polypeptides consisting of 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. The present invention relates to a polypeptide consisting of a combination or all.

(Targeted Delivery) In another embodiment, the present invention provides compositions for targeting cells that express a receptor for a polypeptide of the present invention, or cells that express a cell-associated polypeptide of the present invention. A method of delivering is provided.

As discussed herein, polypeptides or antibodies of the present invention may be hydrophobic,
It can associate with a heterologous polypeptide, heterologous nucleic acid, toxin, or prodrug via hydrophilic, ionic, and / or covalent interactions. In one embodiment, the present invention provides a method for the specific delivery of a composition of the invention to cells by administering a polypeptide of the invention (including an antibody) that associates with a heterologous polypeptide or nucleic acid. I will provide a. In one embodiment, the invention provides a method for delivering a therapeutic protein to a targeted cell. In another embodiment, the invention relates to a single-stranded nucleic acid (eg, an antisense or ribozyme) or a double-stranded nucleic acid (eg,
For example, methods are provided for delivering DNA that can be integrated into the genome of a cell or replicated and transcribed episomally) to targeted cells.

In another embodiment, the present invention provides for identifying a cell by administering a polypeptide of the present invention (eg, a polypeptide of the present invention or an antibody of the present invention) in combination with a toxin or cytotoxic prodrug. Destruction (eg, destruction of tumor cells)
Are provided.

"Toxin" refers to an endogenous cytotoxic effector system, radioisotope,
Compounds that bind and activate holotoxin, modified toxins, catalytic subunits of toxins, or any molecule or enzyme that causes cell death under defined conditions not normally present in or on the cell surface. . Toxins that can be used in accordance with the methods of the present invention include radioisotopes, compounds that bind to an intrinsic or induced endogenous cytotoxic effector system (eg, an antibody (or a complement comprising a portion thereof)) known in the art. Body fixation), thymidine kinase, endonuclease, RNAse, α-toxin, ricin, abrin, Pse
udomonas endotoxin A, diphtheria toxin, saporin, momordin (momo
rdin), gelonin, pokeweed antiviral protein,
Includes, but is not limited to, alpha sarcin and cholera toxin. "Cytotoxic prodrug" refers to an enzyme that normally exists in a cell.
A compound that is converted to a cytotoxic compound and is non-toxic. Cytotoxic prodrugs that can be used in accordance with the methods of the present 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. But not limited to these.

Drug Screening The use of polypeptides of the invention, or polynucleotides encoding these polypeptides, to screen for molecules that alter the activity of the polypeptides of the invention is further contemplated. Such methods include contacting the polypeptides of the present invention with selected compounds suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.

The present invention is particularly useful for screening therapeutic compounds by use of a polypeptide of the present invention, or a binding fragment thereof, in any of a variety of drug screening techniques. The polypeptide or fragment utilized in such a test can be immobilized on a solid support, expressed on the cell surface, free in solution, or localized intracellularly. . 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 a competitive binding assay. For example, the formulation of a conjugate between the drug being tested and the polypeptide of the invention can be measured.

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

Another technique for drug screening provides high-throughput screening for compounds with appropriate binding affinity for the polypeptides of the invention and is disclosed in European Patent Application 84/03564 (September 1984). Published 13th, which is hereby incorporated by reference herein in greater detail. Briefly, large amounts of different small peptide test compounds are synthesized on a solid substrate (eg, a plastic pin or some other surface). A peptide test compound is reacted with a polypeptide of the invention and washed. The bound polypeptide is then detected by methods well known in the art. The purified polypeptide is coated directly on a 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 a polypeptide or fragment thereof. I do. 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.

(Antisense and Ribozymes (Antagonists)) In certain embodiments, the antagonists according to the present invention are of SEQ ID N
O: Nucleic acid sequence corresponding to the sequence contained in 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)). Oligodeoxynu
cleotides as Antisense Inhibitors of
Gene Expression, CRC Press, Boca Rato
n, 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:
560 (1991); Oligodeoxynucleotides as A
ntisense Inhibitors of Gene Expressi
on, CRC Press, Boca Raton, FL (1988). Triple helix formation is described, for example, by Lee et al., Nucleic Acids®.
essearch, 6: 3073 (1979); Cooney et al., Science.
241: 456 (1988); and Dervan et al., Science, 25.
1: 1300 (1991). These methods involve complementary DN
Based on binding of polynucleotide to A or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines has been described previously (Wickstrom (1988); Anfossi et al. (
1989)). These experiments were performed in vitro by incubating cells with oligoribonucleotides. A similar procedure for in vivo applications is described in WO 91/15580. Briefly, a given antisense RN
The oligonucleotide pair for A is generated as follows: a sequence complementary to the first 15 bases of the open reading frame is flanked by a 5 terminal EcoRI site and a 3 terminal HindIII site. Next, the oligonucleotide pair is heated at 90 ° C. for 1 minute and then 2 × ligation buffer (20 mM
Annealed in TRIS HCl pH 7.5, 10 mM MgCl ₂ , 10 mM dithiothreitol (DTT) and 0.2 mM ATP), and then ligated into 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 a mature polypeptide of the present invention. DNA oligonucleotides are designed to be complementary to the region of the gene involved in transcription, thereby inhibiting transcription and production of the receptor. Antisense RNA oligonucleotides hybridize to mRNA in vivo and block translation of the mRNA molecule into receptor polypeptide.

[0648] In one embodiment, the antisense nucleic acids of the invention are produced intracellularly by transcription from a foreign sequence. For example, the vector or a portion thereof is transcribed,
Produce the antisense nucleic acid (RNA) of the present invention. Such a vector contains a sequence encoding the antisense nucleic acid of the invention. Such a vector can remain episomal or integrate chromosomally, as 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. Vectors can be plasmids, viral or others known in the art, used for replication and expression in vertebrate cells. Expression of a sequence encoding a polypeptide of the present invention, or a fragment thereof, may be by any promoter known in the art to work in vertebrate, preferably human cells. Such a promoter can be inducible or constitutive. Such a promoter is SV40
Early promoter region (Bernist and Chambon, Nature
, 29: 304-310 (1981)), the promoter contained in the 3 'long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22: 787-79).
7 (1980)), herpes thymidine promoter (Wagner et al., Proc.
. Natl. Acad. Sci. U. S. A. , 78: 1441-1445 (1
981)), the regulatory sequence of the metallothionein gene (Brinster et al., Nat).
ure, 296: 39-42 (1982)).

The antisense nucleic acids of the present invention include a sequence complementary to at least a portion of the RNA transcript of the gene of interest. However, it is preferred, but not necessary, that they be completely complementary. The sequence "complementary to at least a portion of the RNA" as referred to herein is the sequence R
Means a sequence that is sufficiently complementary to hybridize with NA to form a stable duplex; thus, in the case of the double-stranded antisense nucleic acids of the invention, the duplex D
A single strand of NA 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, the larger the hybridizing nucleic acid, the more base mismatches with the RNA sequences of the invention. And still may form stable duplexes (or even triplexes). One skilled in the art can ascertain the degree of mismatch tolerance by using standard procedures to determine the melting point of the hybridizing complex.

Oligonucleotides that are complementary to the 5 'end of the message (eg, up to and including the AUG start codon) should work most efficiently at inhibiting translation. is there. However, sequences complementary to the 3 'untranslated sequence of the mRNA still had no effect in inhibiting translation of the mRNA. Generally, Wagner, R.A. , Nature, 372: 333-335 (1994). Thus, an oligonucleotide complementary to either the 5'- or 3'-untranslated or non-coding region of a polynucleotide sequence of the present invention will have an endogenous m.
It can be used in antisense approaches to inhibit translation of RNA. mRNA 5
'Oligonucleotides complementary to the untranslated region should include 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 ', 3' or coding regions of the mRNA, the antisense nucleic acid should be at least 6 nucleotides in length, and preferably from 6 to about 50 nucleotides. Oligonucleotides over nucleotide length. In certain aspects, the oligonucleotide comprises at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides.
Nucleotides or at least 50 nucleotides.

The polynucleotides of the present invention may be DNA, or RNA, or chimeric mixtures,
Alternatively, it may be a derivative or modified version, single-stranded, or double-stranded. The oligonucleotide can be modified, for example, with a base moiety, a sugar moiety, or a phosphate backbone to improve molecular stability, hybridization, and the like. The oligonucleotides may be linked to other additional groups such as peptides (eg, to target host cell receptors in vivo) or factors that facilitate transport across cell membranes (eg, Letsinger et al., Proc. Natl. Acad. .Sci
. U. S. A. 86: 6553-6556 (1989); Lemaitre et al.
Proc. Natl. Acad. Sci. 84: 648-652 (1987).
See PCT Publication No. WO 88/09810 (published December 15, 1988)) or the blood-brain barrier (eg, PCT Publication No. WO 89/10134 (
(Published April 25, 1988)), a hybridization-triggered cleavage agent.
gent) (e.g., Krol et al., BioTechniques, 6: 958-).
976 (1988)) or intercalating agents (eg, Zo
n, Pharm. Res. 5: 539-549 (1988)). To this end, the oligonucleotide is a separate molecule (eg, a peptide,
A hybridization-triggering cross-linking agent, a transport agent, a hybridization-triggering cleavage agent, etc.).

The antisense oligonucleotide may include at least one modified base moiety, wherein the 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-galactosylcuosin, 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- Mannosylcuosin, 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.

The antisense oligonucleotide 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, phosphoramidothio Ethates, phosphoramidates, phosphorodiamidates, methylphosphonates, alkyl phosphotriesters, and formacetals or analogs thereof.

[0655] In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. a-anomeric oligonucleotides form specific double-stranded hybrids with complementary RNA, whose 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-methylribonucleotide (Inoue et al., Nucl. Acid)
s Res. 15: 6131-148 (1987)), or chimeric RNA
-A DNA analog (Inoue et al., FEBS Lett. 215: 327).
-330 (1987)).

The polynucleotides of the present invention can be prepared by standard methods known in the art (eg, automated D
With NA synthesizer (such devices are available from Biosearch, Applied B
(commercially available from iosystems, etc.). For example, phosphorothioate oligonucleotides can be prepared using the method of Stein et al. (Nuc).
l. Acids Res. 16: 3209 (1988)).
Methyl phosphonate oligonucleotides are available in controlled pore glass (pore gl).
ass) polymer support (Sarin et al., Proc. Natl. Acad. Sc)
i. U. S. A. 85: 7448-7451 (1988)).

Although antisense nucleotides complementary to the coding region sequences of the present invention can be used, antisense nucleotides complementary to the transcribed untranslated region are most preferred.

[0658] Potential antagonists according to the present invention also include catalytic RNAs, ie, ribozymes (see, eg, PCT Publication WO 90/11364, October 4, 1990).
Sunver et al., Science, 247: 1222-1225 (19
90)). Although a ribozyme that cleaves mRNA at a site-specific recognition sequence can be used to destroy mRNA corresponding to a polynucleotide of the present invention, the use of a hammerhead ribozyme is preferred. The hammerhead ribozyme is located at a position determined by an adjacent region that forms a complementary base pair with the target mRNA.
Cut NA. The only requirement is that the target mRNA has the following two bases: 5'-UG-3 '. The construction and production of hammerhead ribozymes is well known in the art and haseloff and Gerlach
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, it increases efficiency and increases the intracellular accumulation of non-functional mRNA transcripts. Manipulated to minimize.

In the case of an antisense approach, the ribozymes of the invention may be comprised of modified oligonucleotides (eg, having improved stability, targeting, etc.) and express the polynucleotides of the invention in vivo. Should be delivered to cells. A ribozyme-encoding DNA construct can be introduced into cells in the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery is to “encode” a ribozyme under the control of a strong constitutive promoter (eg, such as a pol III or pol II promoter).
Including the use of a DNA construct, the transfected cells produce sufficient amounts of the ribozyme to destroy endogenous messages and inhibit translation. Because ribozymes are catalytic, unlike antisense molecules, lower intracellular concentrations are required for efficiency.

The antagonist / agonist compounds can be used to grow and proliferate the polypeptides of the invention on neoplastic cells and tissues.
feration) effect. That is, it stimulates a tumor agonist,
Thereby delaying or preventing abnormal cell growth and proliferation (eg, in tumorigenesis or proliferation).

[0660] Antagonists / agonists may also be employed to inhibit hypervascular disease and prevent proliferation of epithelial lens cells following extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the invention may also be required, for example, in cases such as restenosis following balloon angioplasty.

The antagonists / agonists may also be utilized to prevent the growth of scar tissue during wound healing.

[0663] Antagonists / agonists may also be utilized to treat the diseases described herein.

Accordingly, the present invention relates to disorders or diseases, including, but not limited to, those disorders or diseases listed throughout this application that are associated with overexpression of a polynucleotide of the present invention ( Methods for treating a patient by administering a) an antisense molecule directed to a polynucleotide of the invention, and / or (b) a ribozyme directed to a polynucleotide of the invention.

Other Activities Polypeptides of the invention are attributed to various disease states (eg, thrombosis, arteriosclerosis, and other cardiovascular conditions) as a result of their ability to stimulate vascular endothelial cell proliferation. May be utilized in procedures to stimulate revascularization of the ischemic tissue. These polypeptides may also be utilized to stimulate angiogenesis and limb remodeling as discussed above.

The polypeptides may also be used in the treatment of wounds resulting from injuries, burns, post-operative tissue repair, and scars. Because they are mitogenic to various cells of different origins (eg, fibroblasts and skeletal muscle cells), and therefore promote the repair or replacement of damaged or diseased tissue.

The polypeptides of the invention also stimulate neuronal growth and treat neuronal damage that occurs in certain neuronal disorders or neurodegenerative conditions (eg, Alzheimer's disease, Parkinson's disease, and AIDS-related complexes). And can be used to prevent. The polypeptides of the present 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 present invention can also be used to prevent aging of the skin due to sunburn by stimulating keratinocyte proliferation.

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

The polypeptides of the present invention can also be utilized to maintain organs prior to transplantation or used to support primordial tissue cell culture.

The polypeptides of the present invention can also be used to derive tissue of mesodermal origin for differentiation in early embryos.

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

The polypeptides or polynucleotides and / or agonists or antagonists of the invention may also have mammalian characteristics (eg, height, weight, hair color,
It can be used to regulate eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (eg, cosmetic surgery). Similarly, the polypeptides or polynucleotides and / or agonists or antagonists of the invention can 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 disorders), tendency to violence, tolerance to pain, fertility (preferably by activin or inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, Or, it can be used to alter the mental or physical state of a mammal by affecting the quality of cognition.

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

Other Preferred Embodiments Another preferred embodiment of the present invention comprises a nucleotide sequence that is at least 95% identical to the sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: X. Includes an isolated nucleic acid molecule, where X is any integer as defined in Table 1.

The sequence of contiguous nucleotides begins at about the nucleotide at position 5 ′ nucleotide of the cloned sequence, as defined for SEQ ID NO: X in Table 1,
Also preferred are nucleic acid molecules that are included in the nucleotide sequence of SEQ ID NO: X, to the extent that they end with a nucleotide approximately at the 3 ′ nucleotide position of the clone sequence.

The sequence of contiguous nucleotides begins approximately at the nucleotide 5 ′ nucleotide of the start codon and approximately 3 ′ nucleotide of the clone sequence as defined for SEQ ID NO: X in Table 1. Nucleic acid molecules that are included in the nucleotide sequence of SEQ ID NO: X, to the extent ending in nucleotides, are also preferred.

The sequence of contiguous nucleotides begins approximately at the nucleotide 5 ′ of the first amino acid of the signal peptide, as defined for SEQ ID NO: X in Table 1, and approximately 3 of the clonal sequence. Nucleic acid molecules, which are included in the nucleotide sequence of SEQ ID NO: X in the range ending with a nucleotide at the 'nucleotide position, are also preferred.

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

[0680] Even more preferred is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO: X.

A further preferred embodiment is a compound as defined for SEQ ID NO: X in Table 1.
Comprises a nucleotide sequence beginning at about the nucleotide 5 'of the first amino acid of the signal peptide and ending at about 3' of the nucleotide sequence of the cloned sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: X 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 are isolated nucleic acid molecules that hybridize to the nucleic acid molecule under stringent hybridization conditions, wherein the hybridizing nucleic acid molecule is an A residue under stringent hybridization conditions. Does not hybridize to nucleic acid molecules having a nucleotide sequence consisting solely or of T 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, wherein the DNA molecule is comprised in a material deposited with the American Type Culture Collection, and The above cDNA clone IDs are given the ATCC accession numbers shown in Table 1.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the 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 included in the ATCC deposit numbered deposit set forth in Table 1.

Also preferred is an isolated nucleic acid molecule wherein the sequence of at least 50 contiguous nucleotides is comprised in the nucleotide sequence of the sequence of the complete open reading frame 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 the sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by the above human cDNA clones.

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

A further preferred embodiment detects in a biological sample a nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: The 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 A nucleotide sequence encoded by a human cDNA clone contained in a deposit having the ATCC accession number indicated for the cDNA clone; the method comprises: selecting a sequence selected from the group described above; Comparing the nucleotide sequence of the nucleic acid molecule with the nucleotide sequence of the nucleic acid molecule; Determining whether the sequence of said nucleic acid molecule in said file is at least 95% identical to said selected sequence.

The step of comparing the sequences comprises determining the degree of nucleic acid hybridization between the nucleic acid molecule in the sample and a nucleic acid molecule comprising the sequence selected from the group above. The above method is also preferred. Similarly, the step of comparing the sequences comprises a nucleotide sequence determined from a nucleic acid molecule in the sample,
The above method, which is performed by comparing with a sequence selected from the above group, is also preferred. Nucleic acid molecules can include DNA 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 sequences 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 of the identified nucleotides: the nucleotide sequence of SEQ ID NO: X, wherein X is And A identified by the cDNA clone ID in Table 1 and shown 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 in the panel 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 in a subject a pathological condition associated with an abnormal structure or expression of a gene encoding a secreted protein identified in Table 1, the method comprising a group consisting of: Detecting a nucleic acid molecule (if any) in a biological sample obtained from the subject, comprising a nucleotide sequence that is at least 95% identical to the sequence of at least 50 contiguous nucleotides in a sequence selected from: Including: the nucleotide sequence of SEQ ID NO: X, where X is any integer as defined in Table 1; and the cDNA clone identified by the cDNA clone ID in Table 1 and described above in Table 1. Encoded by the human cDNA clone contained in the deposit having the ATCC accession number indicated for Nucleotide sequence.

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 is included. Is at least 95% identical to the sequence of at least 50 contiguous nucleotides in a sequence selected from the group above.

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 is Is at least 95% identical to the sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: the nucleotide sequence of SEQ ID NO: X, where X is any integer as defined in Table 1. And the nucleotide sequence encoded by the human cDNA clone identified by the cDNA clone ID in Table 1 and contained in the deposit having the ATCC accession number shown in Table 1 for the cDNA clone above. Nucleic acid molecules can include DNA or RNA molecules.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to the sequence of at least about 10 contiguous amino acids in the amino acid sequence of SEQ ID NO: Y, wherein Y is Any integer as specified.

The sequence of contiguous amino acids begins approximately at the residue at the position of the first amino acid in the secretory moiety, as shown for SEQ ID NO: Y in Table 1, and is approximately the last amino acid in the open reading frame SEQ ID NO: Y in the range ending with the residue
Also preferred are polypeptides comprised in the amino acid sequence of

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

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

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

The cDN identified by the cDNA clone ID in Table 1 and described above in Table 1
Human c contained in the deposit having the ATCC accession number indicated for the A clone
Even more preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to the sequence of at least about 10 contiguous amino acids in the complete amino acid sequence of a secreted protein encoded by a DNA clone.

The above sequence of contiguous amino acids is identified by the cDNA clone ID in Table 1 and is encoded by the human cDNA clone contained in the deposit having the ATCC accession number shown for the above cDNA clone in Table 1. Also preferred are polypeptides comprised in the amino acid sequence of the secretory portion of the secreted protein to be secreted.

The cDN identified by the cDNA clone ID in Table 1 and the cDN in Table 1
An amino acid that is at least 95% identical to the 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, contained in the deposit having the ATCC accession number indicated for the A clone Also preferred is an isolated polypeptide comprising the sequence.

The cDN identified by the cDNA clone ID in Table 1 and
An amino acid that is at least 95% identical to the 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, contained in the deposit having the ATCC accession number indicated for the A clone Also preferred is an isolated polypeptide comprising the sequence.

The cDN identified by the cDNA clone ID in Table 1 and
Human c contained in the deposit having the ATCC accession number indicated for the A clone
Also preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of the secretory portion of the protein encoded by the DNA clone.

An isolated polypeptide 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: Antibodies are further preferred: the amino acid sequence of SEQ ID NO: Y, where Y is any integer defined in Table 1; and identified by the cDNA clone ID in Table 1 and for this cDNA clone in Table 1 Complete amino acid sequence of the protein encoded by the human cDNA clone, contained in a deposit having the indicated ATCC accession number.

A method for detecting in a biological sample a polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: Preferred: the amino acid sequence of SEQ ID NO: Y, where Y is any integer as defined in Table 1; and the ATCC deposit identified by the cDNA clone ID in Table 1 and shown for this cDNA clone in Table 1. The complete amino acid sequence of the protein encoded by the human cDNA clone contained in the numbered deposit; the method compares the amino acid sequence of at least one polypeptide molecule in the sample with a sequence selected from this group. Process and arrangement of the polypeptide molecule in the sample. The sequence has at least 9 sequences of this sequence of at least 10 contiguous amino acids.
Determining whether they are 0% identical.

[0710] The step of comparing the amino acid sequence of at least one polypeptide molecule in the sample to a sequence selected from the group comprises the step of comparing the polypeptides in the sample to a sequence selected from the group consisting 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 at least 10 contiguous amino acids of Is also preferred: the amino acid sequence of SEQ ID NO: Y (where Y
Is any integer defined in Table 1); and the ATC identified by the cDNA clone ID in Table 1 and shown for this cDNA clone in Table 1.
C Complete amino acid sequence of the protein encoded by the human cDNA clone contained in the deposit having the deposit number.

Also preferred is the above method, wherein comparing the sequences is performed by comparing the amino acid sequence determined from the polypeptide molecules in the sample to a sequence selected from this group.

Also preferred is a method of identifying a species, tissue or cell type of a biological sample, wherein the method comprises, if any, at least 10 of the sequences in the sample selected from the group consisting of: Detecting a polypeptide molecule comprising 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 this cDN identified by the cDNA clone ID in Table 1 and in Table 1.
Complete amino acid sequence of a secreted protein, encoded by a human cDNA clone, contained in a deposit having the ATCC accession number indicated for the A clone.

Also preferred is the above method of identifying a species, tissue or cell type of a biological sample, wherein the method comprises detecting a polypeptide molecule comprising an amino acid sequence in a panel of at least two amino acid sequences. And wherein at least one sequence in this panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group above.

[0714] Also preferred is a method of diagnosing in a subject a pathological condition associated with an abnormal structure or expression of a gene encoding a secreted protein identified in Table 1.
The method comprises at least two steps in a biological sample obtained from the subject.
Detecting a polypeptide molecule comprising the amino acid sequence in a panel of two amino acid sequences, wherein at least one sequence in the panel comprises at least 10 contiguous amino acids in a sequence selected from the group consisting of: Is at least 90% identical to the sequence: the amino acid sequence of SEQ ID NO: Y, where Y is any integer as defined in Table 1; and the cDNA identified by the cDNA clone ID in Table 1 and this cDNA in Table 1 Complete amino acid sequence of a secreted protein encoded by a human cDNA clone contained in a deposit having the ATCC accession number indicated for the clone.

[0715] In any of these methods, detecting the polypeptide molecule comprises:
And the use of antibodies.

[0716] 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 contiguous amino acids in a sequence selected from the group consisting of: Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence: 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 shown for this cDNA clone in Table 1.
Complete amino acid sequence of a secreted protein encoded by a human cDNA clone contained in a deposit having a CC accession number.

Also preferred is an isolated nucleic acid molecule wherein the nucleotide sequence encoding the polypeptide has been 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: the amino acid sequence of SEQ ID NO: Y, wherein Y is any of the amino acids defined in Table 1. And the complete amino acid sequence of the secreted protein encoded by the human cDNA clone 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.

[0719] Further preferred is a method of producing a recombinant vector, comprising the step of inserting any of the above isolated nucleic acid molecules into a vector. Recombinant vectors produced by this method are also preferred. Also preferred is a method of producing a recombinant host cell, comprising the step of introducing the vector into a host cell and a recombinant host cell produced by this method.

A method of producing an isolated polypeptide, comprising the steps of culturing the recombinant host cell under conditions such that the polypeptide is expressed, and recovering the polypeptide The method is also preferred. This method of making an isolated polypeptide is also preferred, wherein the recombinant host cell is a eukaryotic cell, and the polypeptide comprises a human comprising an amino acid sequence selected from the group consisting of: Secreted portion of secreted protein: the first of the secreted portion of SEQ ID NO: Y
The amino acid sequence of SEQ ID NO: Y, where Y is an integer set forth in Table 1 and this position of the first amino acid of the secreted portion of SEQ ID NO: And the amino acid of the secreted portion of the protein encoded by the human cDNA clone, which is identified by the cDNA clone ID in Table 1 and is included in the deposit having the ATCC accession number indicated for this cDNA clone in Table 1. Array. Also preferred are isolated polypeptides produced by this method.

[0721] Methods of treating an individual in need of increased levels of secreted protein activity are also preferred. The method includes providing such an individual with a pharmaceutical composition 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.

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

In certain embodiments of the present invention, for each “Contig ID” listed in the fourth column of Table 2, comprises or consists of the nucleotide sequence referenced in the fifth column of Table 2. One or more polynucleotides are preferably excluded and the general formula ab (where a and b are uniquely determined for the corresponding SEQ ID NO: X referred to in column 3 of Table 2) ).
More particular embodiments relate to polynucleotide sequences that exclude one, two, three, four or more particular polynucleotide sequences mentioned in the fifth column of Table 2. This listing is not meant to include all sequences that can be excluded by the general formula, which is merely representative. The references available through these registries are hereby incorporated by reference in their entirety.

[0724]

[Table 2] Having generally described the present invention, the present invention is provided for illustrative purposes,
It can be more easily understood by reference to the following examples, which are not intended to be limiting.

Examples Example 1: Isolation of Selected cDNA Clones from a Deposited Sample Each cDNA clone in the referenced ATCC deposit is contained in a plasmid vector. Table 1 shows 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 shown in Table 1 as being isolated in the vector "Lambda Zap",
The corresponding deposited clone is “pBluescript”.

[0726]

[Table 3] Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636) and Uni-Zap XR (U.S. Pat. No. 5,128,25).
Nos. 5,286,636), Zap Express (U.S. Patent 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)).
ne Cloning Systems, Inc. , 11011 N.R. Torr
eye Pines Road, La Jolla, CA, 92037. pBS contains the ampicillin resistance gene, and pBK contains the neomycin resistance gene. Both are E.I. coli strain XL-1 Blue, which is also available from Stratagene, can be transformed. pBS
Arrives in four forms: SK +, SK-, KS + and KS. S and K are
T7 and T3 primers flanking the polylinker region ("S" is SacI and "K" is KpnI, which are the first sites at each end of each of the linkers) Refers to the orientation of the polylinker with respect to the sequence. "
"+" Or "-" refers to the orientation of the f1 origin of replication ("ori") such that a single-stranded rescue initiated from the f1 ori in one direction produces sense-strand DNA and the other is antisense. Say.

The vectors pSport1, pCMVSport 2.0 and pCMVSpo
rt3.0 from Life Technologies, Inc. , P. O. Box
6009, obtained from Gaithersburg, MD 20897. All Sport vectors contain the ampicillin resistance gene and coli strain D
H10B, which is also available from Life Technologies, can be transformed. (See, eg, Gruber, CE et al., Focus.
15:59 (1993)). Vector rafmid BA (Ben
to Soares, Columbia University, NY) contains an ampicillin resistance gene and E. coli strain XL-1 Blue. Vector pCR® 2.1 (this is Invitroge
n, 1600 Faraday Avenue, Carlsbad, CA 92
008), which contains the ampicillin resistance gene, and c
oli strain DH10B (available from Life Technologies) (eg, Clark, JM, Nuc. Acids).
Res. 16: 9677-9686 (1988) and Mead, D .; Bi
o / Technology 9: (1991)). Preferably, the polynucleotide of the present invention does not include the phage vector sequence identified for a particular clone in Table 1, as well as the corresponding plasmid vector sequence 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 associated with that given clone. Including different cDNA clones)
May be included. Thus, the deposits sharing the same ATCC accession number include at least the plasmid for each cDNA clone shown in Table 1. Typically, Table 1
The sample of each ATCC deposit, cited in, contains a mixture of approximately equal amounts (by weight) of about 50 plasmid DNAs, each containing a different cDNA clone; , More or less than 50 cds
It may contain plasmids for NA clones (up to about 500 cDNA clones).

[0729] Two approaches can be used to isolate a particular clone from the deposited sample of plasmid DNA referred to for that clone in Table 1. First, the plasmid is isolated directly by screening clones using the 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 are treated with, for example, ³² P-γ-ATP
Label with 4 polynucleotide kinase and purify according to conventional methods. (See, for example, Maniatis et al., Molecular Cloning: A.
Laboratory Manual, Cold Spring Harbo
r Press, Cold Spring, NY (1982)). The plasmid mixture can be prepared using techniques known to those of skill in the art (eg, techniques provided by the vector supplier or provided in the relevant publications or patents cited above) using a suitable host as described above. (For example, XL-1 Blue (Strata
gene)). Transformants are plated on 1.5% agar plates (containing the appropriate selection agent, eg, ampicillin) at a density of approximately 150 transformants (colonies) per plate. These plates are prepared using conventional methods for bacterial colony screening (see, eg, Sambrook et al., Mol.
eco Cloning: A Laboratory Manual, 2nd edition, (1989), Cold Spring Harbor Laboratory.
or press, pages 1.93 to 1.104) or other techniques known to those of skill in the art using a nylon membrane.

Alternatively, both ends of SEQ ID NO: X (ie, the 5′N of the clone defined in Table 1)
17 to 2) (in the region of SEQ ID NO: X surrounded by T and 3′NT).
Two primers of zero 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 having 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 dAT each
P, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.2
5 units of Taq polymerase. 35 cycles of PCR (1 minute denaturation at 94 ° C .; 1 minute annealing at 55 ° C .; 1 minute extension at 72 ° C.)
Performed using an rkin-Elmer Cetus automated thermal cycler. The amplification products are analyzed by agarose gel electrophoresis, and DNA bands of the expected molecular weight are cut out and purified. The PCR product is confirmed to be the selected sequence by subcloning and sequencing the DNA product.

Several methods are available for identifying 5 ′ non-coding or 3 ′ non-coding portions of a gene that may not be present in the deposited clone. These methods include, but are not limited to: filter probe search, clonal 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 is available to generate the missing 5 'end of the desired full-length transcript (Frommont-Racine et al., Nucleic A.
cids Res. 21 (7): 1683-1684 (1993)).

[0733] Briefly, a particular RNA oligonucleotide is ligated to the 5 'end of a population of RNA, possibly containing a full-length gene RNA transcript. Using a primer set containing primers specific to the ligated RNA oligonucleotide and primers known to the known sequence of the gene of interest, PCR the 5 'portion of the desired full length gene.
Amplify. The amplified product can then be sequenced and used to generate the full length gene.

The above method starts with total RNA isolated from the desired source, but may use poly A + RNA. The RNA preparation is then treated, if necessary, with a phosphatase to degrade or damage RNA that can interfere with the subsequent RNA ligase step.
The 5 'phosphate group of 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 capped 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. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5 'end using primers specific for the ligated RNA oligonucleotide and primers known for the known sequence of the gene of interest. . The resulting product is then sequenced and analyzed to confirm that the 5 'terminal sequence belongs to the desired gene.

Example 2 Isolation of Genomic Clones Corresponding to Polynucleotides 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 Polypeptides The tissue distribution of mRNA expression of the polynucleotides of the invention was determined, inter alia, by Samb
Determine 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 was replaced with the rediprime ^™ DNA labeling system.
(Amersham Life Science) using, according to the manufacturer's instructions, labeled with P ^32. After labeling, the probe is purified using a CHROMA SPIN-100 ^™ column (Clontech Laboratories, Inc.) according to the manufacturer's protocol number PT1200-1. Various human tissues are then tested for mRNA expression using the purified labeled probe.

Multiple tissue northern (MTN) blots (Clontech) containing various human tissues (H) or human immune system tissues (IM) were prepared using the ExpressHyb ^™ hybridization solution (Clontech) using the manufacturer's protocol number P
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 Chromosomal Mapping of Polynucleotides A set of oligonucleotide primers is designed according to the sequence at the 5 ′ end of SEQ ID NO: X. The primer preferably spans about 100 nucleotides.
This primer set is then used in the 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. Somatic cell hybrid panels containing individual chromosomes or chromosome fragments (Bios, Inc)
In addition, human, mouse, and hamster DNA are used as templates. Reactions are analyzed on either an 8% polyacrylamide gel or a 3.5% agarose gel. Chromosome mapping was performed at approximately 100 in specific somatic cell hybrids.
Determined by the presence of the bp PCR fragment.

Example 5: Bacterial Expression of Polypeptides Polynucleotides encoding the polypeptides of the present invention were constructed using PCR oligos corresponding to the 5 'and 3' ends of the DNA sequence, as outlined in Example 1. Amplification is performed using nucleotide primers to synthesize the insert fragment. Primers used to amplify the cDNA insert should preferably contain restriction sites such as BamHI and XbaI at the 5 'end of the primer to clone the amplification product into an expression vector. For example, BamHI and XbaI are compatible with the bacterial expression vector pQE-9 (Qiagen, Inc., Chatsworth,
CA) corresponding to the restriction enzyme site. This plasmid vector has antibiotic resistance (
Amp ^r ), bacterial origin of replication (ori), IPTG-regulatable promoter /
It encodes an operator (P / O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and a restriction enzyme cloning site.

The pQE-9 vector is digested with BamHI and XbaI, and the amplified fragment is ligated into the pQE-9 vector while maintaining the reading frame initiated in bacterial RBS. The ligation mixture was then transformed into plasmid pRE, which expresses the lacI repressor and confers kanamycin resistance (Kan ^r ).
E.P. E. coli strain M15 / rep4 (Qiagen, I
nc. ). 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.

[0741] Clones containing the desired constructs were selected using Amp (100 μg / ml) and Kan (
Overnight (O / N) in liquid culture in LB medium supplemented with both (25 μg / ml).
Proliferate. The O / N culture is used to inoculate a large culture at a ratio of 1: 100 to 1: 250. Cells were grown at an optical density of 600 (OD) between 0.4 and 0.6.
. Proliferate to ⁶⁰⁰ ). Next, IPTG (isopropyl-BD-thiogalactopyranoside) is added to a final concentration of 1 mM. IPTG is lacI
Induced by inactivation of the repressor, removes P / O obstacles and leads to increased gene expression.

The cells are grown for an additional 3-4 hours. The cells were then centrifuged (6000 ×
g for 20 minutes). The cell pellet is treated with the chaotropic agent 6
Solubilize in M guanidine HCl by stirring at 4 ° C. for 3-4 hours. Cell debris was removed by centrifugation, and the supernatant containing the polypeptide was removed from a nickel-nitrilotriacetic acid ("Ni-NTA") affinity resin column (QI
AGEN, Inc. (Available from the 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 (in particular, The QIAexpressionistion (1
995) QIAGEN, Inc. , See above).

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

The purified protein is then added to phosphate buffered saline (PBS) or 5
Regenerate by dialysis 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 6 M to 1 M urea in 500 mM NaCl, 20% glycerol, 20 mM Tris / HCl pH 7.4, containing protease inhibitors. Regeneration should take at least 1.5 hours. After regeneration, the protein is eluted by adding 250 mM imidazole. Imidazole was added to PBS or 50 mM sodium acetate buffer pH6 and 200 mM N2.
It is removed by a final dialysis step against aCl. Purified protein at 4 ° C
Or freeze at -80 ° C.

In addition to the expression vectors described above, the present invention further includes an expression vector comprising pHE4a and a phage operator and a promoter element operably linked to the polynucleotide of the present invention (ATCC Accession No. 209645).
, Deposited on February 25, 1998). This vector contains: 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
itersburg, MD). Promoter and operator sequences are made synthetically.

The vector is restricted with NdeI and XbaI, BamHI, XhoI, or Asp718, the restriction product is run on a gel, and the larger fragment (the stuffer fragment should be about 310 base pairs) Can be inserted into pHEa. The DNA insert was digested with NdeI (5 ′ primer) and XbaI, BamHI, XhoI,
Alternatively, it is generated according to the PCR protocol described in Example 1 using a PCR primer with a restriction site for Asp718 (3 'primer). PCR
The insert is gel purified and restricted with a compatible enzyme. The insert and the vector are ligated according to standard protocols.

The engineered vector can be easily replaced in the above protocol to express the protein in a bacterial system.

Example 6 Purification of Polypeptides from Inclusion Bodies The following alternative method is used for E. coli when the polypeptide is present in the form of inclusion bodies. c
It can be used to purify polypeptides expressed in oli. Unless otherwise specified, all steps below are performed at 4-10 ° C.

[0750] E. Upon completion of the production phase of the E. coli fermentation, the cell culture was cooled to 4-10 ° C,
And continuous centrifugation at 15,000 rpm (Heraeus Sepatech)
Harvest the cells by Based on the expected yield of protein per unit weight of cell paste and the amount of purified protein required, the appropriate amount (by weight) of cell paste is determined by adding 100 mM Tris, 50 mM EDTA, pH
Suspend in a buffer solution containing 7.4. The cells are dispersed into a homogeneous suspension using a high shear mixer.

The cells were then transferred to a microfluidizer (
Microfluidics, Corp. Or APV Gaulin, Inc
. ) Twice by passing the solution through at 4000-6000 psi.
The homogenate is then mixed with a NaCl solution to a final concentration of 0.5M NaCl, followed by centrifugation at 7000 × g for 15 minutes. The resulting pellet was washed with 0.5 M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.0.
Wash again using 4.

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

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

To clarify the refolded polypeptide solution, a previously 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). Filter the filtered sample with a cation exchange resin (eg, Poros
HS-50, Perseptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0, and eluted in a stepwise fashion with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer. 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 is then combined with a previously prepared strong anion (Poros HQ).
-50, Perceptive Biosystems) exchange resin and weak anion (Poros CM-20, Perceptive Biosystems)
) Load a set of series columns of exchange resin. Equilibrate the column with 40 mM sodium acetate, pH 6.0. Fill both columns with 40 mM sodium acetate,
Wash with 200 mM NaCl, pH 6.0. Next, 10 columns of CM-20 were added.
Linear gradient of column volume (0.2 M NaCl, 50 mM sodium acetate, pH
(6.0-1.0 M NaCl, 50 mM sodium acetate, pH 6.5). Fractions are collected under constant _A280 monitoring of the eluate. The fractions containing the polypeptide (as determined by, for example, 16% SDS-PAGE) are then pooled.

[0756] The resulting polypeptide should show greater than 95% purity after the refolding and purification steps described above. When 5 μg of purified protein is loaded, no major contaminant bands should be observed from the 16% SDS-PAGE gel stained with Coomassie blue. The purified protein also contains endotoxin / LPS
Mixing can be tested and typically the LPS content is less than 0.1 ng / ml according to the LAL assay.

Example 7 Cloning and Expression of a Polypeptide in a Baculovirus Expression System In this example, a polynucleotide is inserted into a baculovirus using the plasmid shuttle vector pA2 to express the polypeptide. This expression vector contains the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by BamHI, X
It contains convenient restriction sites such as baI, and Asp718. The simian virus 40 ("SV40") polyadenylation site is used for efficient polyadenylation. For easy selection of recombinant viruses, the plasmid was transformed into E. coli under the control of a co-oriented weak Drosophila promoter. It contains the β-galactosidase gene from E. coli followed by the polyadenylation signal of the polyhedrin gene. The inserted gene is flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus expressing the cloned polynucleotide. .

[0758] Many other baculovirus vectors (eg, pAc373, pVL941
, And pAcIM1), as will be readily appreciated by those skilled in the art, are signals in which the construct is properly arranged for transcription, translation, secretion, etc. (including signal peptides and in-frame AUGs, if required). Can be used in place of the vector described above. Such vectors are, for example, Lucko
et al., Virology 170: 31-39 (1989).

Specifically, the cDNA sequence contained in the deposited clone, which includes the AUG start codon identified in Table 1 and the naturally associated leader sequence, was replaced by the PCR described in Example 1. Amplify using protocol. If a naturally occurring signal sequence is used to produce a secreted protein, the pA2 vector does not require a second signal peptide. Alternatively, the vector can be transferred to Summers et al.
A Manual of Methods for Baculovirus
Vectors and Insect Cell Culture Proc
edures ", Texas Agricultural Expertimen
tal Station Bulletin No. 1555 (1987)) and can be modified to include a baculovirus leader sequence (pA2 GP).

The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean”, BIO 101 Inc., La Jolla, Ca). The fragment is then digested with the appropriate restriction enzymes and again
Purify on a% agarose gel.

The plasmid can be digested with the corresponding restriction enzymes and, if necessary, dephosphorylated with calf intestinal phosphatase using conventional procedures known in the art.
The DNA was then transferred to a commercially available kit ("Geneclean" BIO 101 In).
c. , La Jolla, Ca) using a 1% agarose gel.

The fragment and the dephosphorylated plasmid are ligated together using T4 DNA ligase. E. FIG. E. coli HB101 or XL-1 Blue (S
Tratagene Cloning Systems, La Jolla, C
a) Other suitable E. coli such as cells. The E. coli host is transformed with the ligation mixture and spread on culture plates. Bacteria containing plasmids are identified by digesting DNA from individual colonies and analyzing the digestion products by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.

[0783] 5 μg of the plasmid containing this polynucleotide was ligated to Felgner et al., Pr.
oc. Natl. Acad. Sci. ScL USA 84: 7413-7417 (19
87) using the lipofection method described by, commercially available linearized baculovirus DNA of 1.0 [mu] g ( "BaculoGold ^TM baculovi
rus DNA ", Pharmingen, San Diego, CA). 1 μg of BaculoGold ^™ viral DNA
And 5 μg of plasmid were added to 50 μl of serum-free Grace's medium (Life Te
channels Inc. , Gaithersburg, MD).
Mix in sterile wells of a microtiter plate. Thereafter, 10 μl of Lipofectin and 90 μl Grace's medium were added, mixed and mixed at room temperature for 15 minutes.
Incubate for minutes. Then, the transfection mixture was applied to Sf9 insect cells (
ATCC CRL 1711). Then plate at 27 ° C. for 5
Incubate for hours. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The culture is then continued at 27 ° C. for 4 days.

After 4 days, supernatants are collected and plaque assays are performed as described by Summers and Smith, supra. "Blue Gal" (Life Tec
hnologies Inc. An agarose gel containing G.I., Gaithersburg) is used to allow easy identification and isolation of clones expressing gal (resulting in blue-colored plaques). (A detailed description of this type of “plaque assay” is also available at Life Technologies Inc.,
(See pages 9-10 in the User's Guide for Insect Cell Culture and Baculovirology distributed by Gaithersburg.) After appropriate incubation, the blue-colored plaque is picked up with a micropipette (eg, Eppendorf) tip. The agar containing the recombinant virus was then resuspended in a microcentrifuge tube containing 200 μl of Grace's medium, and the suspension containing the recombinant baculovirus was seeded on a 35 mm dish of Sf.
Used to infect 9 cells. Four days later, the supernatants of these culture dishes are collected and then stored at 4 ° C.

[0764] To confirm polypeptide expression, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. Cells were infected at a multiplicity of infection of about 2 ("MOI").
), The cells are infected with the recombinant baculovirus containing the polynucleotide. If radiolabeled protein is desired, the medium is removed after 6 hours and SF900 II medium without methionine and cysteine (LifeTechn).
logos Inc. , Rockville, MD). After 42 hours, 5 μCi of ³⁵ S-methionine and 5 μCi of ³⁵ S-cysteine (available from Amersham) are added. The cells are incubated for a further 16 hours and then harvested by centrifugation. The proteins in the supernatant and intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

[0768] Microterminal sequencing of the amino-terminal amino acid sequence of the purified protein can be used to determine the amino-terminal sequence of the produced protein.

Example 8 Expression of a Polypeptide in Mammalian Cells The polypeptide 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 termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences, and intervening sequences flanking donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved using the early and late promoters from SV40, the long terminal repeat (LTR) from retroviruses (eg, RSV, HTLVI, HIVI), and the early promoter of cytomegalovirus (CMV). You. However, cellular elements such as the human actin promoter can also be used.

[0768] Expression vectors suitable for use in practicing the present invention include, for example, pSVL
And pMSG (Pharmacia, Uppsala, Sweden), pR
SVcat (ATCC 37152), pSV2dhfr (ATCC 3714)
6), pBC12MI (ATCC 67109), pCMVSport2.0,
And vectors such as pCMVSport 3.0. Mammalian host cells that can be used include human Hela cells, 293 cells, H9 cells and Jurkat cells, mouse NIH3T3 cells and C127 cells, Cos 1 cells, Cos 7 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 identification and isolation of the transfected cells.

[0770] The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is
It is useful for developing cell lines that carry hundreds or thousands of copies of the gene of interest.
(For example, Alt, FW, et al., J. Biol. Chem. 253: 1357-
1370 (1978); Hamlin, J. M .; L. And Ma, C.I. , Bioch
em. et Biophys. Acta, 1097: 107-143 (1990)
): Page, M .; J. And Sydenham, M .; A. , Biotechn
9: 64-68 (1991)). Another useful selectable marker is the enzyme glutamine synthase (GS) (Murphy et al., Bioc
hem J .; 227: 277-279 (1991); Bebbington et al.
Bio / Technology 10: 169-175 (1992)). Using these markers, mammalian cells are grown in selective media and cells with the highest resistance are selected. These cell lines are integrated into the chromosome,
Contains amplified genes. Chinese hamster ovary (CHO) and NSO
Cells are often used for the production of proteins.

The expression vectors pC4 (ATCC Accession No. 209646) and pC6 (AT), which are derivatives of plasmid pSV2-dhfr (ATCC Accession No. 37146)
CC Accession No. 209647) is due to Rous sarcoma virus (Cullen et al., Mol.
eculular and Cellular Biology, 438-47 (
(March 1985)) and a fragment of the CMV enhancer (Boshart et al., Cell 41: 521-530 (1985)). For example, multiple cloning sites having restriction sites for BamHI, XbaI, and Asp718 facilitate cloning of the gene of interest. The vector also contains the 3 'intron of the rat preproinsulin gene, polyadenylation and termination signals, and the mouse DHFR gene under the control of the SV40 early promoter.

Specifically, for example, plasmid pC6 is digested with an appropriate restriction enzyme,
The calf intestinal phosphatase (phosphph) is then followed according to procedures known in the art.
ate). The vector is then isolated from a 1% agarose gel.

The polynucleotides of the present invention are amplified according to the protocol outlined in Example 1. This vector does not require a second signal peptide if a naturally occurring signal sequence is used to produce the secreted protein. Or,
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).

The amplified fragments were prepared using a commercially available kit (“Geneclean”, BIO 10
1 Inc. , La Jolla, Ca. ) To isolate from a 1% agarose gel. The 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 the 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, for example, using restriction enzyme analysis.

[0776] Chinese hamster ovary cells lacking the active DHFR gene are used for transfection. 5 μg of the expression plasmid pC6 pC4 was transformed with 0.5 μg of the plasmid pSV using lipofectin (Felgner et al., Supra).
Co-transfect with neo. Plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. Cells were treated with 1 mg / ml G4
Seed in α minus MEM supplemented with 18. Two days later, cells were trypsinized and 10, 25, or 50 ng / ml methotrexate and 1 mg / ml
Seed in hybridoma cloning plates (Greiner, Germany) in α-minus MEM supplemented with ml of G418. After about 10-14 days,
Single clones were trypsinized and then different concentrations of methotrexate (5
(0 nM, 100 nM, 200 nM, 400 nM, 800 nM) to seed 6-well petri dishes or 10 ml flasks. The clones growing at the highest concentration of methotrexate were then reduced to higher concentrations (1 μM, 2 μM, 5 μM
(10 mM, 20 mM) in a new 6-well plate containing methotrexate. The same procedure is repeated until a clone is obtained that grows 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 polypeptide of the present invention is preferably fused to another protein. These fusion proteins can be used for various applications. For example, fusion of a polypeptide of the invention to a His tag, an HA tag, a protein A, an IgG domain, and a maltose binding protein facilitates purification (see Example 5; EP A 394,827). Traunecker et al., Nature 331;
: 84-86 (1988)). Similarly, IgG-1, IgG-
3, and fusion to albumin increases half-life in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target proteins to specific subcellular locations. 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 may increase the solubility and / or stability of the fusion protein as compared to a non-fusion protein. All forms of the above fusion proteins can be made by modifying the following protocol, which outlines fusion of polypeptides to IgG molecules, or the protocol described in Example 5.

[0778] Briefly, the human Fc portion of an IgG molecule comprises 5 'and 3' of the sequences described below.
It can be PCR amplified using primers over the ends. These primers should also have convenient restriction sites to facilitate cloning into expression vectors, preferably mammalian expression vectors.

For example, if pC4 (Accession No. 209646) is used, the human Fc portion can be ligated into a BamHI cloning site. Note that the 3 'BamHI site should be destroyed. Next, the vector containing the human Fc portion is re-restricted by BamHI, the vector is linearized, and the polynucleotide of the present invention isolated by the PCR protocol described in Example 1 is ligated into this BamHI site. Is done. Note that this polynucleotide is cloned without a stop codon, otherwise no fusion protein will be 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 may be modified to include a heterologous signal sequence (see, eg, WO 96/34891). Human IgG Fc region

[0781]

Embedded image Example 10 Production of Antibodies from Polypeptides The antibodies of the present invention can be prepared by various methods. (Current Pr
otocols, see Chapter 2). As one example of such a method, cells expressing a polypeptide of the invention are administered to an animal to induce the production of serum containing polyclonal antibodies. In a preferred method, a preparation of a secreted protein is prepared and purified to be substantially free of natural contaminants. Such a preparation is then introduced into an animal to produce a higher specific activity polyclonal antiserum.

In a most preferred method, an antibody of the invention is a monoclonal antibody (or
Its protein binding fragment). Such a monoclonal antibody,
Can be prepared using hybridoma technology (Koehler et al., Nature
256: 495 (1975); Koehler et al., Eur. J. Immuno
l. 6: 511 (1976); Koehler et al., Eur. J. Immunol
. 6: 292 (1976); Hammerling et al .: Monoclonal.
Antibodies and T-Cell Hybridomas, Els
evier, N .; Y. 563-681 (1981)). Generally, such procedures involve immunizing an animal, preferably a mouse, with the polypeptide, or more preferably, with a secretory polypeptide-expressing cell. Such cells can be cultured in any suitable tissue culture medium; however, supplemented with 10% fetal calf serum (inactivated at about 56 ° C.) and about 10 g / l of non-essential amino acids, about 1 Preferably, the cells are cultured in Earle's modified Eagle's medium supplemented with 2,000 U / ml penicillin and about 100 μg / ml streptomycin.

[0783] The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line can be used in accordance with the present invention; however, it is preferred to use the parent myeloma cell line (SP2O) available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then described by Wands et al. (Gastroenterology 80:22).
Cloning by limiting dilution as described in 5-232 (1981)). The hybridoma cells resulting from such a selection are then assayed to identify clones that secrete antibodies capable of binding the polypeptide.

Alternatively, additional antibodies that can bind to the polypeptide can be generated in a two-step procedure using anti-idiotypic antibodies. Such methods take advantage of the fact that antibodies are themselves antigens, and thus it is possible to obtain antibodies that bind to a second antibody. According to this method, using protein-specific antibodies,
An animal (preferably a mouse) is immunized. The spleen cells of such animals are then used to produce hybridoma cells. And these hybridoma cells
The antibody's ability to bind to a protein-specific antibody is screened to identify clones that produce the antibody that can be blocked by the polypeptide. Such antibodies include anti-idiotypic antibodies to protein-specific antibodies,
It can then be used to immunize animals to induce the formation of additional protein-specific antibodies.

[0785] It is clear that Fab and F (ab ') 2 and other fragments of the antibodies of the invention can be used according to 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 technology or by 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 a genetic construct derived from a hybridoma cell producing the monoclonal antibody 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., US Patent No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neube.
rger et al., WO 8615333; Robinson et al., WO 870267.
1; Boulianne et al., Nature 312: 643 (1984); Ne.
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 then be used in the screening assays described in Examples 13-20.

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

293T cells (no cells beyond P + 20) were harvested at 2 × 10 ⁵ cells / well in 0.5 ml of DMEM (Dulbecco modified Eagle's medium) (4.
5 G / L glucose and L-glutamine (12-604F Biowhite)
taker)) / 10% heat-inactivated FBS (14-503F Biowhi)
ttaker) / 1 × Penstrep (17-602E Biowhitta)
ker). Grow cells overnight.

The next day, in a sterile solution container (basin), 300 μl Lipofectamine (1
8324-012 Gibco / BRL) and 5 ml Optimem I (
Mix 31985070 Gibco / BRL) / 96 well plates together. Using a small volume multi-channel pipettor, place about 2 μg of the expression vector containing the polynucleotide insert produced by the method described in Example 8 or 9 into 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 by pipetting up and down gently. 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, the transfection is performed in a team with the following activities (
tag-teaming). By forming a tag team,
The time effort is halved and cells do not spend too much time on PBS. First, A removes the medium from the four 24-well plates of cells, and then B rinses each well with 0.5-1 ml of PBS. Mr. A then aspirated off the PBS rinse, and Mr. B. used a 12-channel pipettor with a tip every other channel and 200 μl of DNA.
The / Lipofectamine / Optimem I complex is added to each row on a 24-well plate, first to odd wells, then to even wells. Incubate at 37 ° C. for 6 hours.

[0792] While incubating the cells, either 1% BSA in DMEM with 1 × penstrep, or CHO-5 medium containing 2 mM glutamine and 1 × penstrep (116.6 mg / L CaCl ₂ (anhydrous Object); 0
. _{00130mg / L CuSO 4 -5H 2 O} ; 0.050mg / L of _{Fe (NO 3) 3 -9H 2} O; 0.417mg / L FeSO 4 of -7H ₂ O; 311.80mg
/ L KCl; 28.64 mg / L MgCl ₂ ; 48.84 mg / L MgS
O _4; 6995.50mg / L of NaCl; 2400.0mg / L NaHCO ₃ in; 62.50mg / L of _{NaH 2 PO 4 -H 2 O;} 71.02mg / L of Na ₂ HP
O _4; the 1.022mg / L cholesterol;; 0.002 mg / L arachidonic _{acid; 0.4320mg / L ZnSO 4 -7H 2} O of 0.070mg / L of 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 palmito oleic acid (palmitric)
acid); 0.010 mg / L palmitic acid; 100 mg / L Pluro
nic F-68; 0.010 mg / L stearic acid; 2.20 mg / L T
ween 80; 4551 mg / L D-glucose; 130.85 mg / ml L-alanine; 147.50 mg / ml L-arginine-HCl; 7.50 m
g / ml of L- asparagine -H ₂ O; 6.65mg / ml of L- aspartic acid; 29.56mg / ml of L- cystine 2HCl-H ₂ O; 31.29mg /
ml of L-cystin-2HCl; 7.35 mg / ml L-glutamic acid; 36
52. L-glutamine at 5.0 mg / ml; glycine at 18.75 mg / ml;
48 mg / ml of L- histidine _{-HCl-H 2 O; 106.97mg /} ml of L- isoleucine; 111.45mg / ml of L- leucine; 163.75Mg
/ Ml L-lysine HCl; 32.34 mg / ml L-methionine; 68.4
8 mg / ml L-phenylalanine; 40.0 mg / ml L-proline; 2
6.25 mg / ml L-serine; 101.05 mg / ml L-threonine;
Of 19.22mg / ml L- tryptophan; 91.79mg / ml of L- tyrosine _{(Tryrosine) -2Na-2H 2 O} ; 99.65mg / ml of L-
Valine; 0.0035 mg / L biotin; 3.24 mg / L D-Ca pantothenic acid; 11.78 mg / L choline chloride; 4.65 mg / L folic acid; 15.6
0 mg / L i-inositol; 3.02 mg / L niacinamide; 3.0
0 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; 5
5.0 mg / L sodium pyruvate; 0.0067 mg / L sodium selenite; 20 μM ethanolamine; 0.122 mg / L ferric citrate; complexed with 41.70 mg / L linoleic acid Methyl-B-cyclodextrin; 33.33 mg / L methyl-B-cyclodextrin complexed with oleic acid; and 10 mg / L retinal complexed with methyl-B-cyclodextrin). Prepare an appropriate medium. (For a 10% BSA stock solution, BSA (81-068-3 Bayer) 1 in 1 L DMEM
00g was dissolved). The medium is filtered and 50 μl are collected in a 15 ml polystyrene conical for the endotoxin assay.

The transfection reaction is terminated at the end of the incubation period, preferably in a tag team. Mr. A aspirates off 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 day 4, 600 μl using a 300 μl multichannel pipettor
Was aliquoted into one 1 ml deep well plate and the remaining supernatant was
Aliquot into the deep wells. Then, the supernatant from each well was prepared in Examples 13-2.
0 can be used in the assays described.

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

Example 12 Construction of GAS Reporter Constructs One signaling pathway involved in cell differentiation and proliferation is the Jaks-ST
Called the AT path. Activated proteins in the Jaks-STAT pathway bind to gamma activation site "GAS" elements or interferon-sensitive response elements ("ISRE"), located in the promoters of many genes.
Binding of the protein to these elements alters the expression of the relevant gene.

The GAS and ISRE elements are recognized by signal transducers and activators of transcription, a class of transcription factors called “STATs”. There are six members of the STAT family. S
tat1 and Stat3, like Stat2 (like a broad response to IFNα), are present in many cell types. Stat4 is more restricted and absent in many cell types, but is found in T helper class I cells after treatment with IL-12. Stat5 was originally called breast growth factor, but 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 represent a unique family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3.
These kinases show significant sequence similarity and are generally catalytically inactive in resting cells.

[0799] Jaks are activated by a wide range of receptors as summarized by the table below. (Schidler and Darnell, Ann. Rev. Bi.
ochem. 64: 621-51 (1995). Jaks
The family of cytokine receptors 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 comprises IFN-a, IFN-g, and IL-
10 inclusive. Class 1 receptors contain a conserved cysteine motif (a set of four conserved cysteines and one tryptophan), and WSXWS
Share a motif (membrane-proximal region encoding Trp-Ser-Xxxx-Trp-Ser (SEQ ID NO: 2)).

Thus, upon binding of the ligand to the receptor, Jaks are activated, which in turn activates STATs, which then migrate and bind to GAS elements. This entire process is involved in the Jaks-STAT signaling pathway.

[0806] Jak therefore reflected by the binding of GAS or ISRE elements
Activation of the s-STAT pathway can be used to indicate proteins involved in cell growth and differentiation. For example, growth factors and cytokines are available from Jaks-S
It is known to activate the TAT pathway. (See table below). Thus, by using a GAS element linked to a reporter molecule, Jak
Activators of the s-STAT pathway can be identified.

[0802]

[Table 4] To construct a synthetic GAS containing the 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 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 adjacent to the XhoI site. The sequence of the 5 'primer is as follows:

[0803]

Embedded image The downstream primer is complementary to the SV40 promoter and
flanking the dIII site: 5 ': GCGGCAAGCTTTTTGCAAAG
CCTAGGC: 3 '(SEQ ID NO: 4).

The 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 / HindIII and subcloned into BLSK2- (Stratagene). Sequencing using forward and reverse primers confirms that the insert contains the following sequence:

[0805]

Embedded image When this GAS promoter element is linked to the SV40 promoter,
Next, the GAS: SEAP2 reporter construct is manipulated. Here, the reporter molecule is secreted alkaline phosphatase, or “SEAP”. However, obviously, in either this or other examples, any reporter molecule can replace SEAP. 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.

The synthetic GAS-SV40 promoter element identified by the above sequence was combined with HindIII and XhoI to create a GAS-SEAP vector.
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 the neomycin resistance gene and is therefore not preferred for mammalian expression systems.

Thus, to generate a stable mammalian cell line that expresses a GAS-SEAP reporter, the GAS-SEAP cassette was removed from the GAS-SEAP vector with SalI and NotI and GAS-SEAP / Neo To create the vector, these restriction sites at the multiple cloning site are used to create a backbone vector containing the neomycin resistance gene (eg, pGFP
-1 (Clontech)). Once the vector has been transfected into mammalian cells, the 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 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 promoters alone or in combination (eg, GAS / NF-KB / EGR, G
AS / NF-KB, Il-2 / NFAT, or NF-KB / GAS). Similarly, other cell lines (eg, HELA (epithelial cells), HUVECs (endothelial cells), Reh (B cells), Saos-2 (osteoblasts), HUVACs (aortic cells), or cardiomyocytes) are 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 identify factors and that supernatants containing polypeptides of the invention proliferate T cells. And / or assessing T cell activity by determining whether to differentiate. The activity of T cells was determined by GA prepared in Example 12.
Evaluate using the S / SEAP / Neo construct. Thus, factors that increase SEAP activity exhibit 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 in Molt-3 cells (ATCC accession number CRL-155).
2) and Molt-4 cells (ATCC accession number CRL-1582) cells may also be used.

[0810] Jurkat T cells are lymphoblastic CD4 + Th1 helper cells. Approximately 2 million Jurkat cells were converted to DMR to create a stable cell line.
GAS-SEAP using IE-C (Life Technologies)
/ 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 genticin are selected. Grow resistant colonies, then
Test their response to increasing concentrations of interferon gamma. 3 shows the dose response of selected clones.

In particular, the following protocol will provide enough cells for 75 wells containing 200 μl of cells. Thus, to produce enough cells for multiple 96-well plates, either this is scaled up or performed in multiples. Jurkat cells were transformed with RPMI + 10% serum and 1% Pen
-Keep 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 for 15-45 minutes at room temperature.

During the incubation time, the cell concentration was counted, the required number of cells (10 ⁷ per transfection) was spun down, and the final concentration was 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 of 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 the polypeptide of the invention and / or induced with the polypeptide of the invention to be produced by the protocol described in Example 11.

The cells should be washed on the day of treatment with the supernatant and 500,000 / ml
It should be resuspended in fresh RPMI + 10% serum to a density of 0 cells. The exact number of cells required will depend on the number of supernatants screened.
Approximately 10 million cells are required for one 96-well plate (100 million cells for 10 plates).

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

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

The 96-well dish containing the Jurkat cells treated with the supernatant is placed in an incubator for 48 hours (note: this time can be varied between 48 and 72 hours)
. 35 μl of sample from each well is then transferred to an opaque 96-well plate using a 12-channel pipette. The opaque plate should be covered (using a cellophane cover) and stored at -20 <0> C until the SEAP assay described in Example 17 is performed. The plate containing the remaining treated cells is placed at 4 ° C. and, if desired, serves as a source of material to repeat the assay on a particular well.

As a positive control, 100 units / ml of interferon gamma may be used, which is known to activate Jurkat T cells. Typically, more than 30-fold induction is observed in the positive control wells.

The above-described protocol can be used to generate both transient and stable transfected cells, which 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 determine whether a polypeptide of the invention will proliferate and / or differentiate myeloid cells. To assess myeloid activity. Myeloid cell activity is assessed using the GAS / SEAP / Neo construct produced in Example 12. Therefore, the factor that increases SEAP activity is Jaks-S
Figure 2 shows the ability to activate the TATS signaling pathway. The myeloid cells used in this assay are U937 (pre-monocytic cell line), but TF-1, HL60, or KG1 may also be used.

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 collected and washed with PBS. U937 cells are usually
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 of GAS-SE
AP2 plasmid DNA, 140 mM NaCl, 5 mM KCl, 375 μM
_{Na 2 HPO 4 · 7H 2 O} , 1mM MgCl 2, and 675μM CaCl ₂
The cells are suspended in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer containing Incubate at 37 ° C. for 45 minutes.

The cells were washed with RPMI 1640 medium containing 10% FBS and then washed with 10% FBS.
Resuspend in ml of complete medium and incubate at 37 ° C for 36 hours.

GAS-SEAP / U937 stable cells are obtained by growing cells in 400 μg / ml G418. G418-free medium is used to grow routinely, but every 1-2 months the cells are grown at 400 μg / ml G418 for two passages.
Should be regrown in.

The cells are tested by collecting 1 × 10 ⁸ cells, which is sufficient for ten 96-well plate assays, and washing with PBS. Suspend cells at a final density of 5 × 10 ⁵ cells / ml in 200 ml of growth medium as described above. 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 interferon gamma can be used, which is known to activate U937 cells. Typically, more than 30-fold induction is observed in positive control wells. The supernatant was purified according to the protocol described in Example 17 by S
Perform EAP assay.

Example 15: High Throughput Screening Assays to Identify Neuronal Activity As cells undergo differentiation and proliferation, a group of genes are activated via 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 a reporter molecule
One promoter can be used to assess cell activation.

[0827] Specifically, the following protocol is used to assess neuronal activity in the PC12 cell line. PC12 cells (rat pheochromosome (phenochrom)
proliferating and / or differentiating by activation with a number of mitogens, such as TPA (tetradecanoylphorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth factor). Is known. Activates EGR1 gene expression during this treatment. Thus, by stably transfecting PC12 cells with a construct containing the EGR promoter linked to a SEAP reporter, activation of PC12 cells can be assessed.

[0827] The EGR / SEAP reporter construct can be assembled according to the following protocol.
EGR-1 promoter sequence (-633 to +1) (Sakamoto K et al., O.
ncogene 6: 867-871 (1991)) can be PCR-amplified from human genomic DNA using the following primers:
3 ′ (SEQ ID NO: 6) 5 ′ GCGAAGCTTCCGCGACTCCCCGGATCCGCTCC-3
'(SEQ ID NO: 7).

[0832] 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
The GAS: SEAP / Neo vector is linearized using indIII and GA
Remove the S / SV40 stuffer. The EGR1 amplification product is subjected to restriction treatment using these same enzymes. Ligate the vector and the EGR1 promoter.

To prepare 96-well plates for cell culture, the coating solution (
Collagen Type I (Upstate Biotech Inc. Cat. No. 08
-115) in 1% dilution with 30% ethanol (sterile filtration)
Add 2 ml per m plate or 50 ml per well in a 96 well plate and air dry for 2 hours.

[0832] PC12 cells were plated on pre-coated 10 cm tissue culture dishes supplemented with 10% horse serum (JRH BIOSCIENCES, Cat. No. 124) supplemented with penicillin (100 units / ml) and streptomycin (100 μg / ml).
49-78P) RPMI-164 with 5% heat-inactivated fetal bovine serum (FBS)
Grow routinely in Medium 0 (Bio Whitaker). A 1 to 4 split is performed every 3 to 4 days. Remove cells from plate by scraping and resuspend by pipetting up and down more than 15 times.

The EGR / SEAP / Neo construct was purified using the Lipofecta 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. G418-free medium is used for conventional growth, but every 1-2 months, cells should be regrown in 300 μg / ml G418 for 2 passages.

To assay for neuronal activity, 10 cm plates with cells at approximately 70-80% confluence are screened by removing old media. The cells are washed once with PBS (phosphate buffered saline). The cells were then transferred to low serum medium (1% horse serum and 0.5% FBS with antibiotics).
In RPMI-1640).

[0832] The next morning, remove the medium and wash the cells with PBS. The cells are scraped off the plate and the cells are suspended in 2 ml of low serum medium. Count the cell number and add lower serum medium to reach a final cell density of 5 × 10 ⁵ cells / ml.

Add 200 μl of the cell suspension to each well of a 96-well plate (1 × 1
0 equal to ⁵ cells / well). 50 μl of the supernatant produced according to Example 11 was
Add at 37 ° C for 48-72 hours. As a positive control, P via EGR
A growth factor known to activate C12 cells (eg, 50 ng / μl nerve growth factor (NGF)) may be used. Typically, more than 50-fold SEAP induction is seen in positive control wells. Supernatant was purified according to Example 17 with SE
Perform AP assay.

Example 16: High Throughput Screening Assay for T Cell Activity NF-KB (nuclear factor KB) has a wide variety of agents, including the inflammatory cytokines IL-1 and TNF, CD30 and CD40. , Lymphotoxin-α and Lymphotoxin-β), by exposure to LPS or thrombin, and by the expression of certain viral gene products. As transcription factors, NF-KB is the expression of genes involved in immune cell activation, regulates apoptosis (NF-KB appears to protect cells from apoptosis),
Regulates B and T cell development, antiviral and antimicrobial responses, and multiple stress responses.

In unstimulated conditions, NF-KB is retained in the cytoplasm with I-KB (inhibitor KB). However, upon stimulation, I-KB is phosphorylated and degraded, causing a shuttle of NF-KB to the nucleus, thereby activating transcription of the target gene. Target genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1
MHC.

[0839] Due to its central role and ability to respond to a range of stimuli, NF-
A reporter construct utilizing the KB promoter element is used to screen the supernatant produced in Example 11. Activators or inhibitors of NF-KB are useful for treating disease. For example, an inhibitor of NF-KB may be used to treat a disease associated with acute or chronic NF-KB activation (eg, rheumatoid arthritis).

To construct a vector containing the NF-KB promoter element, the PCR
Is used. The upstream primer has an NF-KB binding site (
GGGGACTTTCCC) (SEQ ID NO: 8), containing an 18 bp sequence complementary to the 5 'end of the SV40 early promoter sequence and flanking the XhoI site: 5': GCGGCCTCGAGGGGACTTTTCCCGGGGACTTCCC
GGGGACTTCCGGGACTTTCCATCCTGGCCATCTCAA
TTAG: 3 '(SEQ ID NO: 9).

[0841] The downstream primer is complementary to the 3 'end of the SV40 promoter,
And adjacent to the HindIII site: 5 ': GCGGCAAGCTTTTTCAAAGCCTAGGC: 3' (SEQ ID NO: 4).

[0841] 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
Sequencing with the three primers confirms that the insert contains the following sequence:

[0843]

Embedded image The SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) is then replaced with this NF-KB / SV40 fragment using XhoI and HindIII. However, this vector does not contain the neomycin resistance gene and is therefore not preferred for mammalian expression systems.

[0844] To generate a stable mammalian cell line, the NF-KB / SV40 / SEAP cassette was transformed with the NF-KB / SEA described above using the restriction enzymes SalI and NotI.
Remove from P vector and insert into vector containing neomycin resistance.
Specifically, after restriction treatment of pGFP-1 with SalI and NotI, NF-
The KB / SV40 / SEAP cassette was inserted into pGFP-1 (Clontech) to replace the GFP gene.

Once the NF-KB / SV40 / SEAP / Neo vector has been prepared, stable Jurkat T cells are prepared 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 5-10 fold activation is observed.

Example 17: Assay for SEAP activity As a reporter molecule for the assays described in Examples 13-16, SE
AP activity was determined according to the following general procedure for Tropix Phospho-li.
Assay using ht Kit (Catalog number BP-400). Trop
The ix Phospho-light Kit is a dilution buffer used below,
Supply assay buffer and reaction buffer.

The dispenser is filled with 2.5 × dilution buffer and 15 μl of 2.5 × dilution buffer is dispensed into Optiplate containing 35 μl of supernatant. Seal the plate with a plastic sealer and incubate at 65 ° C. for 30 minutes. Separate the Optiplate to avoid uneven heating.

The sample is cooled to room temperature for 15 minutes. Empty the dispenser and fill with assay buffer. Add 50 ml assay buffer and incubate at room temperature for 5 minutes. Empty the dispenser and fill with reaction buffer (see table below). Add 50 μl reaction buffer and incubate for 20 minutes at room temperature. Since the intensity of the chemiluminescent signal is time-dependent and it takes about 10 minutes to read 5 plates on a luminometer, process 5 plates at a time and start a second set after 10 minutes Should be.

Read the relative light unit in the luminometer. Set H12 as blank and print the result. An increase in chemiluminescence indicates a reporter activity.

[0850]

[Table 5] Example 18: High Throughput Screening Assay to Identify Changes in Small Molecule Concentration and Membrane Permeability Binding of a ligand to a receptor alters intracellular levels of small molecules such as calcium, potassium, and sodium and pH. , As well as changing the membrane potential are known. These changes can be measured in assays that identify supernatants that bind to receptors on particular 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 I can do it.

The following assays measure changes in fluorescent molecules (Molecular Probes) that bind small molecules using a fluorimetric imaging plate reader (“FLIPR”). Obviously, any fluorescent molecule that detects small molecules may be a calcium fluorescent molecule, fluo-4 (Molecular Probes,
Inc. Catalog number F-14202).

For adherent cells, cells are seeded at 10,000-20,000 cells / well in clear bottom Co-star black 96-well plates. Plate CO ₂
Incubate for 20 hours in the incubator. The adherent cells were washed in a Biotek washer with 200 μl HBSS (Hank's Balanced Salt).
Solution), leaving 100 μl of buffer after the last wash.

[0853] A stock solution of 1 mg / ml fluo-4 was added to 10% pluronic acid (puro
nic acid) Made with 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 four 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 x 10 ⁶ cells / ml with HBSS in a ml conical tube. Add 4 μl of 1 mg / ml fluo-4 10% pluronic acid in DMSO per ml of cell suspension. The tubes were then placed in a 37 ° C water bath for 30-6.
Leave for 0 minutes. Wash cells twice with HBSS and resuspend to 1 × 10 ⁶ cells / ml,
Then, 100 μl / well is distributed to the microplate. 100 plates
Centrifuge at 0 rpm for 5 minutes. Next, place the plate on Denley CellWa
After washing once with 200 μl in sh, the final volume is brought to 100 μl by aspiration step.

For non-cell based assays, each well contains a fluorescent molecule such as fluo-4. The supernatant is added to the wells and a change in fluorescence is detected.

To measure the fluorescence of intracellular calcium, 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) emission is 530 nm; and (6)
Sample addition is 50 μl. An increase in emission at 530 nm indicates an extracellular signaling event that results in an increase in intracellular Ca ⁺⁺ concentration.

Example 19: High Throughput Screening Assay to Identify Tyrosine Kinase Activity Protein tyrosine kinases (PTKs) represent a diverse group of transmembrane and cytoplasmic kinases. Within the receptor protein tyrosine kinase (RPTK) family there is a range of receptors for mitogenic and metabolic growth factors, including PDGF, FGF, EGF, NGF, HGF and the insulin receptor subfamily. In addition, there is a large RPTK family for which the corresponding ligand is unknown. RPTK ligands include mainly secreted low molecular weight proteins, but also include membrane-bound and extracellular matrix proteins.

[0838] Activation of RPTK by ligand involves ligand-mediated receptor dimerization, which results in transphosphorylation of the receptor subunit and activation of cytoplasmic tyrosine kinase. Cytoplasmic tyrosine kinases include the src family (
For example, receptor-related tyrosine kinases of src, yes, lck, lyn, fyn), and non-receptor-bound and cytosolic protein tyrosine kinases such as the Jak family. These members are members of the cytokine superfamily (eg, interleukins, interferons, GM
-CSF and leptin).

Because of the wide range of known factors that can stimulate tyrosine kinase activity, the identification of these novel human secreted proteins that can activate tyrosine kinase signaling pathways is of interest. Thus, the following protocol is designed to identify novel human secreted proteins that can activate the tyrosine kinase signaling pathway.

[0981] Target cells (eg, primary keratinocytes) were purchased from Nalge Nunc (Naperville, Ill.) At a density of about 25,000 cells / well.
Inoculate well Loprodyne Silent Screen Plate. The plate is sterilized by rinsing twice with 100% ethanol for 30 minutes, rinsing with water and drying overnight. Some plates were prepared with 100 ml of cell culture grade type I collagen (50 mg / ml), gelatin (2%) or polylysine (
50 mg / ml) (all of which are available from Sigma Chemicals (St. L.)
oois, MO) or Becton Dickinso
n (Bedford, MA), coated with 10% Matrigel or calf serum for 2 hours, rinsed with PBS, and stored at 4 ° C. Seed 5,000 cells / well in growth medium and use the manufacturer's Alamar Biosie
nces, Inc. As described by (Sacramento, CA), ala
Cell proliferation on these plates is assayed by indirect quantification of cell numbers after 48 hours using marBlue. Becton Dickinson
(Falcon plate cover # 3071 from Bedford, MA)
Cover Loprodyne Silent Screen Plate. Fal
Con Microtest III cell culture plates can also be used in some growth experiments.

To prepare the extract, A431 cells are seeded on Loprodyne plate nylon membrane (20,000 / 200 ml / well) and cultured overnight in complete medium. Cells are incubated in serum-free basal medium for 24 hours and allowed to rest. 5 to 50 μl of EGF (60 ng / ml) or the supernatant generated in Example 11
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. Next, the plate was removed from the vacuum transfer manifold (vacuum transfer manifold).
ld) and extract the extract using house vacuum at the bottom of each well.
. Filter through a 45 mm membrane. The extracts are collected in a 96-well capture / assay plate at the bottom of the vacuum manifold and immediately placed on ice. To obtain an extract clarified by centrifugation, the contents of each well are taken out after solubilization with a surfactant for 5 minutes, and centrifuged at 4 ° C., 16,000 × g for 15 minutes.

The filtered extract is tested for the level of tyrosine kinase activity. Although a number of methods for detecting tyrosine kinase activity are known, one of the methods is described herein.

Generally, the tyrosine kinase activity of the supernatant is assessed by determining its ability to phosphorylate tyrosine residues on a particular substrate (biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (cytokinase kinase cd)
c2-p34, corresponding to amino acids 6-20) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a series of tyrosine kinases and are available from Boehringer Mannheim.

A tyrosine kinase reaction is set up by adding the following components in order. First, after adding 10 μl of 5 μM biotinylated peptide, the 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 of BSA, 5 μl of sodium vanadate (1 mM) and finally 5 μl of water. The components are mixed gently and the reaction mixture is pre-incubated for 2 minutes at 30 ° C. The reaction is started by adding 10 μl of control enzyme or filtered supernatant.

[0873] The tyrosine kinase assay reaction is then stopped by adding 10 μl of 120 mM EDTA, and the reaction is placed on ice.

[0866] 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. Thereby, streptavidin (streptava)
din) Associate coated 96-well plate with biotinylated peptide.
Wash MTP module four times with 300 μl / well PBS. Next, anti-phosphotyrosine conjugated to horseradish peroxidase.
sine) 75 μl of antibody (anti-P-Tyr-POD (0.5 u / ml)) is added to each well and incubated at 37 ° C. for 1 hour. Wash wells as described 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 was quantified using an ELISA reader, which reflects the level of tyrosine kinase activity.

Example 20: High Throughput Screening Assays to Identify Phosphorylation Activity [0868] Primary cell as a potential alternative and / or complement of the protein tyrosine kinase activity assay described in Example 19 Assays that detect activation (phosphorylation) of internal signaling intermediates may also be used.
For example, as described below, one particular assay is Erk-1 and Erk-2.
Tyrosine phosphorylation of the kinase can be detected. However, Raf, JNK, p38M
Phosphorylation of AP, Map kinase kinase (MEK), MEK kinase, Src, muscle-specific kinase (MuSK), other molecules such as IRAK, Tec and Janus, and any other phosphoserine, phosphotyrosine or phosphothreonine molecule. , Erk-1 or Er with these molecules in the following assays
It can be detected by replacing k-2.

[0868] Specifically, the wells of a 96-well ELISA plate were filled with protein G (1 μg /
Make assay plate by coating with 0.1 ml for 2 hours at room temperature (RT). The plates are then rinsed with PBS and blocked with 3% BSA / PBS for 1 hour at RT. Next, protein G plates were loaded with Erk-1 and Erk-1.
Two commercially available monoclonal antibodies against rk-2 (100 ng / well) (S
Treat (1 hour at RT) with ant Cruz Biotechnology. (This step can be easily modified by replacing the monoclonal antibody that detects any of the above molecules to detect other molecules). After rinsing 3-5 times with PBS, plates are stored at 4 ° C. until use.

A431 cells are seeded at 20,000 / well in 96-well Loprodyne filter plates and cultured overnight in growth medium. Next, the cells were placed in a basal medium (D
After starvation in MEM) for 48 hours, EGF (6 ng / well) or Example 1
Treat with 50 μl of the supernatant obtained in 1 for 5-20 minutes. The cells are then solubilized and the extract is filtered directly into the assay plate.

After incubating with the extract for 1 hour at RT, the wells are rinsed again. As a positive control, a commercially available MAP kinase preparation (10 ng / well)
Is used instead of the A431 extract. The plates were then plated with Erk-1 and Erk-1.
Treatment with a commercially available polyclonal (rabbit) antibody (1 μg / ml) that specifically recognizes the phosphorylated epitope of rk-2 kinase (1 hour at RT). The antibody is biotinylated by standard procedures. Next, the conjugated polyclonal antibody was combined with europium streptavidin and europium fluorescence enhancer and Wallac DELF.
Quantification is by continuous incubation (time-resolved fluorescence) in an IA device. An increase in the fluorescent signal above background indicates phosphorylation.

Example 21 Methods for Determining Alterations in Genes Corresponding to a Polynucleotide [0887] RNA isolated from an entire family or individual patients presenting a phenotype of interest (eg, a disease) is isolated. Next, cDNA is generated from these RNA samples using protocols known in the art (see Sambrook).
Next, this cDNA is used as a template for PCR using primers surrounding the target region in SEQ ID NO: X. Suggested PCR conditions are Sid
ransky, D .; 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 subjected to SequTherm Polymerase (Epi
and sequencing using primers labeled at the 5 'end with T4 polynucleotide kinase. The intron-exon boundaries of the selected exons are also determined and the genomic PCR products are analyzed to confirm the results. Next, the PCR product with the suspected mutation is cloned and sequenced to confirm the direct sequencing results.

The PCR product was prepared according to the method of Holton, T .; A. And Graham, M .; W. , Nu
cleic Acids Research, 19: 1156 (1991), cloned into a T-tail vector, and T7 polymerase (United
(States Biochemical). Affected individuals are identified by mutations that are not present in unaffected individuals.

[0887] Genomic rearrangements are also observed as a way to determine alterations in the gene corresponding to the polynucleotide. Genomic clones isolated according to Example 2 were isolated from digoxigenin deoxy-uridine 5'-triphosphate (Boehringer Manh).
nick) and using Johnson, Cg
. Et al., Methods Cell Biol. 35: 73-99 (1991)
Perform FISH as described in (2). Hybridization with labeled probes is performed using a large excess of human cot-1 DNA for specific hybridization to the corresponding genomic locus.

[0876] Chromosomes are counterstained with 4,6-diamino-2-phenylidol and propidium iodide to generate a combination of C and R bands. Aligned images for accurate mapping were obtained using a triple band filter set (Chroma T).
technology, Brattleboro, VT) and cooled charge-coupled device cameras (Photometrics, Tucson, AZ) and tunable excitation wavelength filters (Johnson, Cv., et al., Genet. Anal. Tech. App.).
l. , 8:75 (1991)). Isee Gra
physical Program System (Invision Cor.
Image collection, analysis and chromosome segment length measurements are performed using the method of the present invention (portation, Durham, NC). Chromosomal changes in the genomic region to which the probe hybridized 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 a Polypeptide in a Biological Sample A polypeptide of the invention can be detected in a biological sample, and an increase or decrease in polypeptide levels is detected. If so, the polypeptide is a marker of a particular phenotype. It is understood that there are a number of detection methods and, therefore, those skilled in the art can modify the following assays to suit their particular needs.

[0887] For example, an antibody sandwich ELISA is used to detect polypeptides in a sample, preferably a biological sample. The wells of the microtiter plate are coated with the specific antibody 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 Example 10. The wells are blocked so that non-specific binding of the polypeptide to the wells is reduced.

[0877] The coated wells are then incubated with the polypeptide-containing sample at RT for more than 2 hours. Preferably, serial dilutions of the sample should be used to confirm the results. The plate is then washed three times with deionized or distilled water to remove unbound polypeptide.

Next, 50 μl of the specific antibody-alkaline phosphatase conjugate was added to 25 to 400 n.
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.

Add 75 μl of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate for 1 hour at room temperature. The reaction is measured with a microtiter plate reader. A standard curve is generated using serial dilutions of control samples, and the polypeptide concentration is plotted on the X-axis (log scale) and the fluorescence or absorbance on the Y-axis (linear scale). Interpolate the polypeptide concentration in the sample using the standard curve.

Example 23: Formulations The present invention also provides administration of an effective amount of a therapeutic agent to a subject, such as a disease or disorder, such as any one or more of the diseases disclosed herein. ) Is provided. The therapeutic agent is a pharmaceutically acceptable carrier type (
For example, a polynucleotide or polypeptide of the invention (including fragments and variants), agonists or antagonists thereof, and / or antibodies thereto, in combination with a sterile carrier).

Therapeutic agents can be determined by taking into account the individual patient's clinical condition (particularly the side effects of the secreted polypeptide alone treatment), site of delivery, method of administration, dosing regimen and other factors known to those of skill in the art. (Good medical practice). Therefore, the "effective amount" intended in the present specification is:
It is determined in consideration of such considerations.

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 the patient's body weight,
As noted above, this is left to therapeutic discretion. More preferably, for this hormone, the 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 a human. When administered continuously, typically the therapeutic agent is administered from about 1 μg / kg / hour to about 50 μg / kg.
Administered either by injection 1 to 4 times daily or by continuous subcutaneous infusion (eg using a minipump) at a dosing rate of / hour. Intravenous bag solutions may also be used. The duration of treatment needed to observe the change and the post-treatment interval at which the response occurs
It seems to vary depending on the desired effect.

Therapeutic agents can be administered orally, rectally, parenterally, intracistemally.
y), administered intravaginally, intraperitoneally, topically (such as by powder, ointment, gel, drops, or transdermal patch), buccally or orally or as a nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of formulation auxiliary. The term "parenteral" as used herein refers to modes of administration, including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0888] Therapeutic agents of the invention are also suitably administered by sustained release systems. The therapeutic agent is
Oral, rectal, parenteral, intracistemally, vaginal,
Intraperitoneal, topical (such as with powders, ointments, gels, drops, or transdermal patches),
It is administered orally or orally or as a nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of formulation auxiliary. The term "parenteral" as used herein refers to modes of administration, including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0887] Therapeutic agents of the invention are also suitably administered by sustained release systems. Suitable examples of sustained release therapeutic agents include suitable polymeric substances (eg, semipermeable polymer matrices in the form of shaped articles (eg, films or microcapsules)), suitable hydrophobic substances (eg, in acceptable quality oils). Or an ion exchange resin, and poorly soluble derivatives (eg, poorly soluble salts).

The sustained release matrix includes 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).

[0889] Sustained-release therapeutics also include compositions of the present invention encapsulated in liposomes (generally, Langer, Science 249: 1527-1533 (1990).
); Treat et al., Liposomes in the Therapy of
Infectious Disease and Censor, Lopez
-Berestein and Fidler (eds.), Liss, New Yo
rk, 317-327 and 353-365 (1989)). Liposomes containing a therapeutic agent can be prepared by methods known per se:
DE 3,218,121; Epstein et al., Proc. Natl. Acad.
Sci. USA 82: 3688-3692 (1985); Hwang et al., Pr.
oc. Natl. Acad. Sci. USA 77: 4030-4034 (19
80); EP52,322; EP36,676; EP88,046; EP143.
, 949; EP 142,641; Japanese Patent Application No. 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545;
02,324. Typically, liposomes are small (about 200-800 °) monolayers, where the lipid content is greater than about 30 mol% cholesterol and the selected proportion is adjusted for optimal therapeutic agents. You.

In a still further embodiment, a Therapeutic of the Invention is delivered by a pump (Langer, supra; Sefton, CRC Crit. Ref. Biome).
d. Eng. 14: 201 (1987); Buchwald et al., Surgery.
88: 507 (1980); Saudek et al. Engl. J. Med. 3
21: 574 (1989)).

Other controlled release systems are described in Langer (Science 249: 1527-15).
33 (1990)).

For parenteral administration, in one embodiment, generally, the therapeutic agent will be administered to a recipient in a desired degree of purity, in a pharmaceutically acceptable carrier, ie, the dosage and concentration employed. It is formulated by mixing it with a non-toxic, non-toxic and compatible with the other ingredients of the formulation in a unit dosage injectable form (solution, suspension or emulsion). For example, the formulation is preferably free of oxidizing agents and other compounds known to be harmful to therapeutic agents.

In general, the formulations are prepared by uniformly and intimately contacting the therapeutic agent with a liquid carrier or a finely divided solid carrier 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. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes.

[0894] The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are non-toxic to recipients at the dosages and concentrations employed, and include such substances as phosphate, citrate, succinate, acetic acid and other organic acids or salts thereof. Antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides (eg, polyarginine or tripeptide); proteins such as serum albumin, gelatin or immunoglobulins; polyvinylpyrrolidone 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 A sugar alcohol such as mannitol or sorbitol Lumpur; counterions such as sodium; and / or polysorbate include nonionic surfactants such as poloxamers or PEG.

Therapeutic agents will typically comprise from about 0.1 mg / ml to 100 mg / ml, preferably 1 mg / ml.
Formulated in such vehicles at a concentration of 〜１０10 mg / ml and a pH of about 3-8. It is understood that the use of the particular excipients, carriers or stabilizers described above results in the formation of polypeptide salts.

[0887] Any therapeutic agent used for therapeutic administration can be sterile. Sterility is readily achieved by filtration through sterile filtration membranes (eg, a 0.2 micron membrane). Generally, the therapeutic agent will be placed in a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Therapeutic agents are typically stored in unit or multi-dose containers, for example, sealed ampules or vials, as an aqueous solution or a lyophilized formulation for reconstitution.
As an example of a lyophilized formulation, sterile filtered 1% (w / v) into 10 ml vials
) Fill 5 ml of aqueous polypeptide solution and freeze-dry the resulting mixture.
The lyophilized polypeptide 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 a therapeutic of the present invention. A notice in the form of a government agency that regulates the manufacture, use or sale of pharmaceuticals or biological products may accompany such containers, and may be issued by the government regarding manufacture, use or sale for human administration. Represents institutional approval. In addition, therapeutic agents may be used in combination with other therapeutic compounds.

[0899] Therapeutic agents of the invention can be administered alone or in combination with an adjuvant. Adjuvants that can be administered with the therapeutics of the invention include, but are not limited to, alum, alum and deoxycholic acid (ImmunoAg), MTP-PE (Biocine Corp.), QS2.
1 (Genentech, Inc.), BCG, and MPL. In certain embodiments, a Therapeutic of the invention is administered in combination with alum. In another specific embodiment, a Therapeutic Agent of the Invention is administered in combination with QS21. Additional adjuvants that can be administered with the therapeutics of the invention include, but are not limited to: monophosphoryl lipid immunomodulators, Adj
uVax 100a, QS-21, QS-18, CRL1005, aluminum salt, MF-59, and Virosomal adjuvant technology. Vaccines that can be administered with the therapeutics of the invention include, but are not limited to: MMR (measles, mumps, rubella), polio, varicella, tetanus / diphtheria, A Hepatitis, hepatitis B, influenza B, pertussis (w
A vaccine directed at protection against hoping cough, pneumonia, influenza, Lyme disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. The combination may be administered either concomitantly (eg, as a mixture, separately but simultaneously or in parallel); or sequentially. This includes a presentation in which the combined agents are administered together as a therapeutic mixture, and the combined agents are administered separately but simultaneously (eg, through separate intravenous lines to the same individual). Procedures are also included. "Combination" administration further includes separately administering one of the compounds or agents given first, followed by the second.

Therapeutic agents of the invention (Therapeutic) 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 Conventional immunotherapeutic agents, cytokines and / or growth factors. The combination may be administered, for example, either simultaneously, as a mixture, separately but simultaneously or concurrently; or sequentially. This includes presentations where the combined agents are administered together as a therapeutic mixture, and also procedures where the combined agents are administered separately but simultaneously (eg, through separate intravenous lines to the same individual). Also includes. "Combination" administration further includes separately administering one of the compounds or agents given first, followed by the second. 1
In one embodiment, a therapeutic of the invention is administered in combination with a member of the TNF family. TNF molecule, TNF that can be administered with a therapeutic of the invention
Related or TNF-like molecules include, but are not limited to: soluble forms of TNF-α, lymphotoxin-α, (LT-α, TNF-
), LT-β (found in the complex heterotrimer LT-α2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1
BBL, DcR3, OX40L, TNF-γ (International publication number WO96 / 1432)
8), AIM-I (International Publication Number WO97 / 33899), Endokine (en)
dokine) -α (International Publication No. WO98 / 07880), TR6 (International Publication No. WO98 / 30694), OPG, and Neutrokine (neutro)
kine) -α (WO 98/18921), OX40, and nerve growth factor (NGF), as well as soluble forms of Fas, soluble forms of CD30, soluble forms of CD27, soluble forms of CD40, and soluble forms of 4-IBB , T
R2 (International publication number WO96 / 34095), DR3 (International publication number WO97 /
33904), DR4 (International publication number WO98 / 32856), TR5 (International publication number WO98 / 30693), TR6 (International publication number WO98 / 30694)
, TR7 (International Publication No. WO98 / 41629), TRANK, TR9 (International Publication No. WO98 / 56892), TR10 (International Publication No. WO98 / 54202)
), 312C2 (WO 98/06842), and TR12, and soluble forms of CD154, soluble forms of CD70, and soluble forms of CD
153.

In certain embodiments, the therapeutic agents of the invention are administered in combination with an antiretroviral agent, a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor and / or a protease inhibitor. Nucleoside reverse transcriptase inhibitors that can be administered in combination with the therapeutic agents of the invention include the following,
Not limited to: RETROVIR ^™ (Zidovudine / AZT), VIDE
Z ^™ (didanocine / ddI), HIVID ^™ (zarcitabine / ddC), ZER
IT ^™ (stavudine / d4T), EPIVIR ^™ (lamivudine / 3TC), and COMBIVIR ^™ (zidovudine / lamivudine). Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the therapeutic agents of the present invention include, but are not limited to: VIRAMUNE ^™ (nevirapine), RESCRI
PTOR ^™ (delavirdine) and SUSTIVA ^™ (efavirenz). Protease inhibitors that can be administered in combination with the therapeutic agents of the present invention include:
Examples include, but are not limited to: CRIXIVAN ^™ (indinavir), NORVIR ^™ (ritonavir), INVIRASE ^™ (saquinavir)
And VIRACEPT ^™ (Nelfinavir). 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 preventing or treating HIV infection. In addition, it can be used in any combination with the therapeutic agent of the present invention.

In another embodiment, a Therapeutic Agent of the Invention may be administered in combination with an anti-opportunistic infection agent. Anti-opportunistic infection agents that can be administered in combination with the therapeutic agents of the present invention include, but are not limited to: TRIMETHOP
RIM-SULFAMETHOXAZOLE ^™ , DAPSON ^™ , PENTA
MIDINE ^™ , ATOVAQONE ^™ , ISONIAZID ^™ , RIFAM
PIN ^TM , PYRAZINAMIDE ^TM , ETHAMBUTOL ^TM , RIFAB
UTIN ^™ , CLARITROMYCIN ^™ , AZITTHROMYCIN ^™ ,
GANICLOVIR ^™ , FOSCARNET ^™ , CIDOFOVIR ^™ , F
LUCONAZOLE ^™ , ITRACONAZOLE ^™ , KETOCONAZO
LE ^™ , ACYCLOVIR ^™ , FAMCICOLVIR ^™ , PYRIMETH
AMINE ^™ , LEUCOVORIN ^™ , NEUPOGEN ^™ (filgrastim / G-CSF), and LEUKINE ^™ (sargramostine (sargra)
most) / GM-CSF). In certain embodiments, a therapeutic agent of the invention is used to prevent or prevent an opportunistic Pneumocystis carinii pneumonia infection in TRIMETHOPRIM-SULFAMETHOXAZ.
OLE ^™ , DAPSONE ^™ , PENTAMIDINE ^™ , and / or AT
Used in any combination with OVAQUAONE ^™ . In another specific embodiment, the therapeutic agent of the present invention is administered with ISONIAZID ^™ , RIFA to prevent or prevent opportunistic Mycobacterium avium complex infection.
MPIN ^™ , PYRAZINAMIDE ^™ , and / or ETHAMBUTO
As used in any combination with L ^TM. In another specific embodiment, the therapeutic agent of the invention is used to prophylactically treat or prevent opportunistic Mycobacterium tuberculosis infection, RIFABUTIN ^™ , CLARIT.
As used in any combination with HROMYCIN ^TM, and / or Azithromycin ^TM. In another specific embodiment, a Therapeutic of the Invention comprises a GANCI to treat or prevent an opportunistic cytomegalovirus infection prophylactically.
Used in any combination with CLIVIR ^™ , FOSCARNET ^™ , and / or CIDOFOVIR ^™ . In another specific embodiment, the therapeutic agent of the invention is used to prophylactically treat or prevent opportunistic fungal infections.
ZOLE ^™ , ITRACONAZOLE ^™ , and / or KETOCONAZ
Used in any combination with OLE ^™ . In another specific embodiment, the therapeutic agent of the invention comprises ACYCLOVIR ^™ and / or FA for the prophylactic treatment or prevention of opportunistic herpes simplex virus type I and / or II infections.
Used in any combination with MCICOLVIR ^™ . In another specific embodiment, the therapeutic agent of the present invention comprises PYRIMETHAMINE ^™ and / or PYRIMETHAMINE ^™ for prophylactically treating or preventing an opportunistic Toxoplasma gondii infection.
Or used in any combination with LEUCOVORIN ^™ . In another specific embodiment, the therapeutics of the invention are used in any combination with LEUCOVORIN ^™ and / or NEUPOGEN ^™ to preventively treat or prevent opportunistic bacterial infections.

In a further embodiment, a Therapeutic Agent of the Invention is administered in combination with an antiviral agent. Antiviral agents that can be administered with the therapeutics of the invention include acyclovir, ribavirin, amantadine, and remantidine (remantidine).
tidine), but is not limited thereto.

In a further embodiment, a Therapeutic of the Invention is administered with an antibiotic. Antibiotics that can be administered with the therapeutics of the invention include amoxicillin, β
Lactamase, aminoglycoside, β-lactam (glycopeptide), β-lactamase, clindamycin, chloramphenicol, cephalosporin, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones,
Examples include, but are not limited to, macrolide antibiotics, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracycline, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.

Conventional non-specific immunosuppressants that can be administered with the therapeutic agents of the present invention include steroids, cyclosporine, cyclosporin analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspell Guarin (15-deoxyspergualin), and response T
Examples include, but are not limited to, other immunosuppressants that act by inhibiting the function of cells.

In certain embodiments, a Therapeutic Agent of the Invention is administered in combination with an immunosuppressant. Immunosuppressant preparations that can be administered with the therapeutics of the invention include ORT
^{HOCLONE TM (OKT3), SANDIMMUNE TM} / NEORAL TM / S
ANGDYA ^™ (cyclosporine), PROGRAF ^™ (tacrolimus), CE
Examples include, but are not limited to, LLCEPT ^™ (mycophenolate), azathioprine, glucocorticosteroids, and RAPAMUNE ^™ (sirolimus). In certain embodiments, immunosuppressive agents can be used to prevent rejection of organ or bone marrow transplants.

In a further embodiment, a Therapeutic Agent of the Invention is 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 GAMMER ^™ , IV
Examples include, but are not limited to, EEGAM ^™ , SANDOGLOBULIN ^™ , GAMMAGARD S / D ^™ and GAMIMUNE ^™ . In certain embodiments, a Therapeutic of the invention is administered in combination with an intravenous immunoglobulin preparation in transplantation therapy (eg, bone marrow transplantation).

In a further embodiment, a Therapeutic Agent of the Invention is 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 glucocorticoid and non-steroidal anti-inflammatory agents, aminoaryl carboxylic acid derivatives, aryl acetic acid derivatives, aryl butyric acid derivatives, aryl carboxylic acids, aryl propionic acid derivatives, pyrazoles, Pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine
e), bendazac, benzidamine, bucolome, difenpyramide, ditazole, emorfazone, guaiazulene, nabumetone, nimesulide, orgothein, oxaseprol, paranyline, perisoxal,
Pifoxime, procazone, proxazole, and tenidap,
It is not limited to these.

[0909] In another embodiment, a composition of the present invention is administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that can be administered with the therapeutic agents of the invention include antibiotic derivatives (eg, doxorubicin, bleomycin, daunorubicin, and dactinomycin); anti-estrogens (eg, tamoxifen); antimetabolites (eg, fluorouracil, 5 -FU, methotrexate, floxuridine, interferon α-2b, glutamic acid, plicamycin
, 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, diethylstilbestrol diphosphate, chlorotrianisene, and test lactone); Nitrogen mustard derivatives (eg, mephalen, chlorambucil, mechlorethamine (ni Roger emissions mustard) and thiotepa); steroids and combinations (e.g., betamethasone sodium); and others (
Examples include, but are not limited to, dicarbazine, asparaginase, mitotene, vincristine sulfate, vinblastine sulfate, and etoposide.

In certain embodiments, a Therapeutic Agent of the invention is administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), or in any combination of the components of CHOP. In another embodiment, a Therapeutic Agent of the Invention is administered in combination with Rituximab. In a further embodiment, the therapeutic of the invention comprises Rituxmab and CHOP
Or with any combination of components of Rituxmab and CHOP.

[0911] In a further embodiment, a Therapeutic of the Invention is administered in combination with a cytokine. Cytokines that can be administered with the therapeutic agents of the present invention include IL
2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13
, IL15, anti-CD40, CD40L, IFN-γ and TNF-α. In another embodiment, the therapeutic agent of the present invention may comprise 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-1
1, IL-12, IL-13, IL-14, IL-15, IL-16, IL-1
7, including, but not limited to, IL-18, IL-19, IL-20 and IL-21).

In a further embodiment, a 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 invention include glioma-derived growth factor (GDGF) as disclosed in European Patent No. EP-399816; platelet-derived as disclosed in European Patent No. EP-682110. Growth Factor A (PDGF-A); Platelet-Derived Growth Factor B (PDGF-B) as disclosed in European Patent No. EP-282317; International Publication No. WO 92/0
Placental Growth Factor (PIGF) as disclosed in US Pat.
Orthh Factors, 4: 259-268 (1993); placental growth factor 2 (PIGF-2); vascular endothelial growth factor (VEGF) as disclosed in International Publication No. WO 90/13649; European Patent No.EP-5064
Vascular endothelial growth factor A (VEGF-A) as disclosed in International Publication No. W;
Vascular endothelial growth factor 2 (VEGF-2) as disclosed in O96 / 39515
Vascular endothelial growth factor B (VEGF-3); vascular endothelial growth factor B-186 (VEGF-B186) as disclosed in International Publication No. WO 96/26736; as disclosed in International Publication No. WO 98/02543. Vascular endothelial growth factor D (VE
GF-D); vascular endothelial growth factor D (VEGF-D) as disclosed in International Publication No. WO 98/07732; and vascular endothelial growth factor E (VEGF-E) as disclosed in German Patent No. DE19639601. ), But is not limited thereto. The above references are incorporated herein by reference.

[0913] In a further embodiment, a Therapeutic of the Invention is administered in combination with a hematopoietic growth factor. Hematopoietic growth factors that can be administered with the therapeutic agents of the present invention include LEU
KINE ^™ (SARGRAMOSTIM ^™ ) and NEUPOGEN ^™ (FIL
GRASTIM ^™ ), but is not limited thereto.

[0914] In a further embodiment, a Therapeutic of the Invention is administered in combination with a 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 FG.
F-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11,
Examples include, but are not limited to, FGF-12, FGF-13, FGF-14, and FGF-15.

In a further embodiment, a Therapeutic Agent of the Invention is administered in combination with another treatment or prophylactic regime (eg, radiation therapy).

Example 24 Method of Treating a Reduced Level of a Polypeptide The present invention relates to a method for treating an individual in need of increasing the level of a polypeptide of the present invention in the body, the method comprising: Administering to such an individual a composition comprising a therapeutically effective amount of an agonist of the present invention (including a polypeptide of the present invention). It is further understood that conditions caused by reduced levels of normal or normal expression of secreted proteins in an individual can be treated by administering a polypeptide of the present invention, preferably in a secreted form. Accordingly, the invention also provides a method of treating an individual in need of increasing levels of this polypeptide. The method includes administering to such an individual an amount of a therapeutic agent comprising the polypeptide that increases the level of activity of the polypeptide in such an individual.

For example, a patient with reduced levels of a polypeptide takes the polypeptide at a daily dose of 0.1-100 μg / kg for six consecutive days. Preferably, the polypeptide is in a secreted form. The exact details of the dosage regimen based on administration and formulation can be found at:
Provided in Example 23.

Example 25: Methods of Treating Elevated Polypeptide Levels The present invention also relates to a method for treating an individual in need of decreasing the level of a polypeptide of the present invention in the body, the method comprising: Administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of the invention (including a polypeptide and an antibody of the invention).

In one example, antisense technology is used to inhibit the production of a polypeptide of the invention. This technique is one example of a method of reducing the levels of polypeptides (preferably secreted forms) resulting from various etiologies such as cancer. For example, patients diagnosed with abnormally elevated levels of polypeptide may receive 0.5, 1.0, 1.5, 2.0 and 3.0 mg of antisense polynucleotide.
Iv / kg / day for 21 days. If the treatment is well tolerated, the treatment is repeated after a 7 day washout period. The formulation of this antisense polynucleotide is provided in Example 23.

Example 26 Treatment Methods Using Gene Therapy-Ex Vivo One method of gene therapy transplants fibroblasts capable of expressing a polypeptide into a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in a tissue culture medium and divided into small pieces. A small chunk of tissue is placed on the wet surface of a tissue culture flask and approximately 10 pieces are placed in each flask. The flask is inverted, closed tightly, and left overnight at room temperature. 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,
Ham's F12 containing 10% FBS, penicillin and streptomycin
Medium). The flask is then incubated at 37 ° C. for about one week.

At this point, fresh medium is added and then changed every few days. After another two weeks of culture, a monolayer of fibroblasts appears. This monolayer is trypsinized,
Scale up to a larger flask.

[0922] The Moloney murine sarcoma virus long terminal repeat is flanked by pMV-7 (Kirsc
hmeier, P .; T. Et al., DNA, 7: 219-25 (1988)).
After digestion with RI and HindIII, it is treated with calf intestinal phosphatase.
The linear vector is fractionated on an agarose gel and purified using glass beads.

[0923] The cDNAs encoding the polypeptides of the present invention may be ligated to the 5 'and 3' end sequences as described in Example 1, respectively, using primers and having appropriate restriction sites and start / stop codons, if necessary. Can be amplified using PCR primers corresponding to Preferably, the 5 'primer contains an EcoRI site and the 3' primer contains a HindIII site. Equal amounts of the Moloney murine sarcoma virus linear skeleton and the amplified EcoRI and HindIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for joining the two fragments. The ligation mixture is then used to transform bacteria HB101. Next, it is plated on agar containing kanamycin to confirm that the vector has the gene of interest correctly inserted.

[0924] Amphotropic pA317 or GP + am12 packaging cells were
Grow to confluent density in tissue culture in Dulbecco's Modified Eagle's Medium (DMEM) containing 0% calf serum (CS), penicillin and streptomycin. Next, the MSV vector containing the gene of interest is added to the medium, and the packaging cells are transduced with the vector. At this time, the packaging cells produce infectious virus particles containing the gene (here, the packaging cells are referred to as producer cells).

[0925] Fresh media is added to the transduced producer cells, and the media is then harvested from 10 cm plates of confluent producer cells. The used medium containing the infectious virus particles is filtered through a Millipore filter to remove the detached producer cells before using this medium to infect fibroblasts. Remove the medium from the subconfluent plate of fibroblasts and immediately replace with the medium from the producer cells. Remove this medium and replace with fresh medium. If the titer of the virus is high, virtually all fibroblasts will be 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.

[0926] The engineered fibroblasts are then implanted into a host, either alone or after being grown to confluence on cytodex 3 microcarrier beads.

Example 27 Gene Therapy Using an Endogenous Gene Corresponding to a Polynucleotide of the Invention Another method of gene therapy according to the present invention is directed to the use of an endogenous polynucleotide sequence of the present invention, for example, in US Pat. No. 5,641,670 (issued June 24, 1997);
International Publication No. WO 96/29411 (published Sep. 26, 1996); International Publication No. WO 94/12650 (published Aug. 4, 1994); Koller et al., P.
rc. Natl. Acad. Sci. USA, 86: 8932-8935 (1
989); and Zijlstra et al., Nature, 342: 435-438.
(1989) operably linked to a promoter via homologous recombination. The method involves the activation of a gene that is present in the target cell, but is not expressed or expressed at a lower level than desired in the cell.

[0928] A polynucleotide construct is generated that includes a promoter and a targeting sequence. This target sequence is homologous to the 5 'non-coding sequence of the endogenous polynucleotide sequence, adjacent to the promoter. The targeting sequence is 5 'of the polynucleotide sequence.
Close enough to the ends so that the promoter is operably linked to the endogenous sequence during homologous recombination. This promoter and targeting sequence are
Can be used to amplify. Preferably, the amplified promoter contains different restriction sites on the 5 'and 3' ends. Preferably, the 3 'end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter, and the 5 'end of the second targeting sequence is the 3' end of the amplified promoter. And 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 the digested targeting sequence are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of these two fragments. The 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 a transfection facilitating agent (eg, liposomes, viral sequences, viral particles, precipitants, etc.). Such methods of delivery are known in the art.

[0931] Once the cells are 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 the polynucleotide in the cell. Expression can be detected by immunological staining or any other method known in the art.

[0932] Fibroblasts are obtained from a subject by skin biopsy. The obtained tissue was used for DMEM +
Place in 10% fetal calf serum. Fibroblasts in the logarithmic or early stationary phase are trypsinized and rinsed with nutrient media from the surface of the plastic. An aliquot of the cell suspension is removed for counting and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is washed with 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM K
(Cl, 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.

[0932] 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 Xbal 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. This CMV promoter and these fragments (1 and 2) were replaced with the appropriate enzymes (CMV promoter-XbaI and BamHI; fragment 1-XbaI; fragment 2-B
amHI) and ligated together. The resulting ligation product is converted to HindII
Digested with I and ligated with the pUC18 plasmid digested with HindIII.

The plasmid DNA was transferred to a sterile cuvette with a 0.4 cm electrode gap (B
io-Rad). The final DNA concentration is generally at least 120μ
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 device (Bio-Rad). The capacitance and voltage are set to 960 μF and 250-300 V, respectively. Increasing the voltage decreases cell viability, but dramatically increases the percentage of viable cells that stably integrate the introduced DNA into their genome. Given these parameters, a pulse time of about 14 to 2
0 mSec should be observed.

The electroporated cells are maintained at room temperature for about 5 minutes, and the contents of the cuvette are then gently removed using a sterile transfer pipette. The cells were placed in a pre-warmed nutrient medium (D with 15% bovine serum) in a 10 cm dish.
(MEM) add directly to 10 ml and incubate at 37 ° C. The next day, the medium was aspirated and replaced with 10 ml of fresh medium and an additional 16-24
Incubate for hours.

[0932] The engineered fibroblasts are then injected into the host, either alone or after being grown to confluence on cytodex3 microcarrier beads. I do. here,
The fibroblasts produce a protein product. The fibroblasts can then be introduced into a patient as described above.

Example 28: Methods of Treatment Using Gene Therapy-In Vivo Another aspect of the present invention is the use of in vivo gene therapy methods to treat disorders, diseases, and conditions. This method of gene therapy involves the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into animals to increase or decrease expression of the polypeptide. 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. Techniques and methods for such gene therapy and delivery are known in the art and are described, for example, in WO 90/11.
092, WO 98/11779; U.S. Patent Nos. 5,693,622 and 5,7051,
No. 5,558,059; Tabata et al., Cardiovasc. Re
s. 35 (3): 470-479 (1997); Chao et al., Pharmaco.
l. Res. 35 (6): 517-522 (1997); Wolff, New.
omuscul. Disord. 7 (5): 314-318 (1997), Sc
hwartz et al., Gene Ther. 3 (5): 405-411 (1996)
); Tsurumi et al., Circulation 94 (12): 3281-3.
290 (1996), incorporated herein by reference.

[0938] The polynucleotide constructs are delivered by any method that delivers an injectable substance to the cells of an animal, such as injection into the interstitial space of tissue (heart, muscle, skin, lung, liver, intestine, etc.). obtain. The polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to any delivery vehicle (viral sequence, viral particle, liposome formulation, lipofectin, or (Including a precipitant and the like). However, the polynucleotides of the present invention can also be prepared in liposome formulations (eg, Felgner), which 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. (1995) Biol. Cell 85 (
1): 1-7).

[0941] The polynucleotide vector constructs used in the gene therapy method include:
Preferred are constructs that do not integrate into the host genome and do not contain sequences that allow replication. Any strong promoter known to those of skill in the art can be used to drive expression of the DNA. Unlike other gene therapy techniques, one major advantage of introducing a naked nucleic acid sequence into a target cell is the transient nature of polynucleotide synthesis in that cell. 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 six months.

[0941] This polynucleotide construct can be used in tissues (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, eyes, glands, and connective tissue) Can be delivered to the space. The interstitial space of this tissue may be between the intercellular fluid, mucopolysaccharide matrix (reticulated fibers of organ tissue, elastic fibers in the walls of blood vessels or chambers, collagen fibers of fibrous tissue), or connective tissue sheath Includes the same matrix in sexual muscle cells or in bone hiatus. This is also the space occupied by the circulating plasma and the lymph of the lymph channels. Delivery of muscle tissue to the interstitial space is preferred for reasons discussed below. They can be conveniently delivered by injection into the tissue containing these cells. They are preferably delivered to and expressed in differentiated, persistent, non-dividing cells, but are delivered and expressed in non-differentiated cells or in fully differentiated cells (eg, blood stem cells). Or dermal fibroblasts). In vivo, muscle cells are particularly competent in their ability to take up and express polynucleotides.

For naked polynucleotide injection, effective dosages of DNA or RNA range from about 0.05 g / kg to about 50 mg / kg body weight. Preferably, the dosage will be from about 0.005 mg / kg to about 20 mg / kg, and more preferably, from about 0.05 mg / kg to about 5 mg / kg. Of course, as the skilled artisan will appreciate, this dosage will vary according to the tissue site of the injection. Appropriate and effective dosages of nucleic acid sequences can be readily determined by one skilled in the art, and will depend on the condition being treated and the route of administration. The preferred route of administration is by parenteral injection into the interstitial space of the tissue. However, other parenteral routes can also be used,
This includes, for example, inhalation of an aerosol formulation, especially for delivery to the lung or bronchial tissue, the throat, or the mucous membrane of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in this procedure.

[0938] The dose response effect of the injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for the production of mRNA encoding a polypeptide of the present invention is prepared according to standard recombinant DNA methodology. Mold DN
A, which can be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps of the mouse are then injected with various amounts of template DNA.

[0978] Female and male Balb / C mice, 5-6 weeks old, were given 0.3 ml of 2.5%
Anesthetize by injecting Avertin intraperitoneally. A 1.5 cm incision is made in the anterior thigh and the quadriceps are visualized directly. The template DNA is injected in a 0.1 ml carrier through a 27-gauge needle with a 1 cc syringe for 1 minute into the knee at about 0.5 cm from the distal insertion site of the muscle at a depth of about 0.2 cm. . Sutures are made over the injection site for future positioning and the skin is closed with a stainless steel clip.

After an appropriate incubation time (eg, 7 days), a muscle extract is prepared by cutting out all four animals. Every 15 μm section of each quadriceps muscle is histochemically stained for protein expression. A time course for protein expression can be performed in a similar manner, except that four animals from different mice are harvested 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 supernatant from injected and control mice. The results of the above experiment in mice were
NA can be used to extrapolate appropriate dosages and other treatment parameters in humans and other animals.

Example 29: Transgenic animals The polypeptides of the present invention can also be expressed in transgenic animals. Mouse, rat, rabbit, hamster, guinea pig, pig, mini pig, goat,
Animals of any species, including but not limited to sheep, cattle and non-human primates (eg, baboons, monkeys and chimpanzees) can be used to make transgenic animals. In certain embodiments, the techniques described herein or otherwise known in the art are used for expression of a polypeptide of the invention in humans as part of a gene therapy protocol.

[0947] Any technique known in the art for introducing a transgene (ie, a polynucleotide of the present invention) into an animal can be used to create a founder strain (fou) of the transgenic animal.
ndr line). Such techniques are described in Pronuclear Microinjection (Paterson et al., Appl. Microbiol. Biotec).
hnol. 40: 691-698 (1994); Carver et al., Biotec.
hnology (NY) 11: 1263-1270 (1993); Wright
Et al., Biotechnology (NY) 9: 830-834 (1991); and Hoppe et al., US Pat. No. 4,873,191 (1989); Retrovirus-mediated gene transfer into the germline (Van der Putten et al., Pr.
oc. Natl. Acad. Sci. USA 82: 6148-6152 (19
85)), blastocyst or embryo; 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 the polynucleotide of the present invention using a gene gun (see, for example, Ulmer et al., Science 259: 1745 (1993)); into embryonic pluripotent stem cells Introduction of nucleic acid constructs and transfer of this stem cell to blastocysts; and sperm-mediated gene transfer (La
vitrano et al., Cell 57: 717-723 (1989)); and the like. For a review of such techniques, Gordon, "Transgenic An," incorporated herein by reference in its entirety.
imals ", Intl. Rev .. Cytol. 115: 171-229 (19
89).

[0948] Any technique known in the art can be used to generate transgenic clones containing the polynucleotides of the present invention (eg, cultured, quiescently induced embryonic, fetal or adult cells). Nuclear transfer into the nucleus of a cell-derived enucleated oocyte (Campell et al., Nature 380: 64-66 (1996); Will)
Mut et al., Nature 385: 810-813 (1997)).

The present invention provides transgenic animals that have the transgene in all of their cells, as well as animals that have the transgene in some (but not all) cells (ie, mosaic animals or chimeras). I do. The transgene can be a single transgene or a concatamer (eg, head-to-head tandem or head-to-head).
Tail tandem). This transgene can also be obtained, for example, from the teachings of Lasko et al. (Lasko et al., Proc. Natl. Ac.
ad. Sci. USA 89: 6322-2236 (1992)) and can be selectively introduced into specific cell types and activated. The regulatory sequences required for such cell type-specific activation will depend on the particular cell type of interest, and they 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. Briefly, when such a technique is used, a vector containing several nucleotide sequences homologous to the endogenous gene is transformed into a nucleotide sequence of the endogenous gene through homologous recombination with a chromosomal sequence. And designed to disrupt the function of the nucleotide sequence. The transgene can also be selectively introduced into a particular cell type, thus, for example, by Gu et al. (Gu et al., Science 26
5: 103-106 (1994)), and endogenous genes are 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 they will be apparent to those skilled in the art.

[0950] Once the transgenic animal is 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, and analysis of animal tissue can confirm that transgene integration has occurred. Transgene mRNA expression levels in tissues of transgenic animals can also be determined by Northern blot analysis, in situ hybridization analysis and reverse transcriptase P analysis of tissue samples obtained from the animals.
It can be assessed using techniques including, but not limited to, CR (rt-PCR). Samples of the transgenic gene-expressing tissue can also be assessed immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

[0981] Once founders are generated, they can be crossed, inbred, outbred or crossed to produce colonies of a particular animal. Examples of such mating strategies include, but are not limited to: outcrossing of founders with more than one integration site to establish another lineage; additive expression of each transgene By effect, inbreeding of separate strains to produce complex transgenics expressing higher levels of the transgene; certain routines for both enhancing expression and eliminating the need to screen animals by DNA analysis. Crosses of heterozygous transgenic animals that produce animals homozygous for the integration site; crosses of separate homozygous lines to generate compound heterozygous or homozygous lines; and the desired experimental model Crosses to place the transgene on a suitable different background.

[0952] The transgenic animals of the invention can be used to elaborate the biological functions of the polypeptides of the invention, study conditions and / or disorders associated with abnormal expression, and ameliorate such conditions and / or disorders. And has uses including, but not limited to, animal model systems useful for screening for compounds that are effective against.

Example 30: Knockout animals Endogenous gene expression can also be achieved by using targeted homologous recombination to generate genes and / or
Alternatively, it can be reduced by inactivating or "knocking out" the 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, non-functional polynucleotide (or completely unrelated DNA) of the present invention flanked by DNA homologous to an endogenous polynucleotide sequence (either the coding or regulatory region of this gene). Sequence) can be used with or without a selectable and / or negative selectable marker to transfect cells expressing a polypeptide of the invention in vivo. In another embodiment, techniques known in the art are used to create knockouts in cells that contain, but do not express, the gene of interest. Insertion of this DNA construct via targeted homologous recombination results in inactivation of the targeted gene. Such an approach is particularly suitable for the research and agricultural fields that can be used to generate progeny of animals with targeted genes in which modifications to embryonic stem cells are inactive (eg, Thomas and Capecchi 1987 and Thompson 1989). , See above). However, this approach is routine for use in humans when the recombinant DNA construct is directly administered or targeted in vivo at the required site using appropriate viral vectors apparent to those of skill in the art. Can be adapted.

[0954] In a further embodiment of the present invention, a cell engineered to express a polypeptide of the invention, or a cell engineered to not express a polypeptide of the invention (eg, a knockout), Administered to the patient in vivo. Such cells can be obtained from patients (ie, animals including humans) or MHC compatible donors, and include fibroblasts, bone marrow cells, blood cells (eg, lymphocytes), adipocytes, muscle cells, endothelial cells, and the like. But not limited to them. The cells can be transformed, for example, by 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.). (Including, but not limited to, use) to introduce a coding sequence for a polypeptide of the invention into a cell, or to bind to the coding sequence and / or a polypeptide of the invention. To destroy the array,
Genetically engineered in vitro using recombinant DNA technology. The coding sequence for a 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 a polypeptide of the present invention can be introduced systemically into a patient, for example, in the circulation or intraperitoneally.

Alternatively, the cells can be incorporated into a 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 lymphoid or vascular graft (eg, Anderson et al., US Pat. No. 5,399,349 and Mu
See lligan and Wilson, U.S. Patent No. 5,460,959. Each of these is hereby incorporated by reference in its entirety).

If the cells to be administered are non-autologous or non-MHC compatible cells, they may be administered using well-known techniques that would prevent the generation of a host immune response against the transduced cells. For example, the cells can be introduced in a capsular form, which does not allow the introduced cells to be recognized by the host immune system, while allowing the components to exchange with the immediate extracellular environment.

[0957] The transgenic and "knock-out" animals of the invention can be used to describe the biological function of the polypeptides of the invention, study conditions and / or disorders associated with aberrant expression, and to study such conditions and / or disorders. Or has uses including but not limited to animal model systems useful in screening for compounds effective in ameliorating the disorder.

Example 31 Production of Antibodies a. Hybridoma Technology The antibodies of the present invention can be prepared by various methods. (Current Pr
otocols, see Chapter 2). As one example of such a method, cells expressing a polypeptide of the invention are administered to an animal to induce the production of serum containing polyclonal antibodies. In a preferred method, a preparation of a polypeptide of the invention is prepared and purified to be substantially free of natural contaminants. Such a preparation is then introduced into an animal to produce a higher specific activity polyclonal antiserum.

[0981] Monoclonal antibodies specific for a polypeptide of the invention are prepared using hybridoma technology (Kohler et al., Nature 256: 495).
(1975); Kohler et al., Eur. J. Immunol. 6: 511 (1
976); Kohler et al., Eur. J. Immunol. 6: 292 (197)
6); Hammerling et al .: Monoclonal Antibodies.
and T-Cell Hybridomas, Elsevier, N.W. Y.
563-681 (1981)). Generally, an animal, preferably a mouse, is immunized with a polypeptide of the invention, or more preferably, with cells expressing a secreted polypeptide. Such polypeptide-expressing cells are preferably supplemented with 10% fetal calf serum (inactivated at about 56 ° C.) in any suitable tissue culture medium, and contain about 10 g / l of non-essential amino acids, about 1 Cultured in Earle's modified Eagle's medium supplemented with 2,000 U / ml penicillin, and about 100 μg / ml streptomycin.

[0961] The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line can be used according to the present invention;
It is preferred to use the parental myeloma cell line (SP2O) available from CC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then are described in Wands et al. (Gastroenterology 80: 225-232 (
1981)) by cloning by limiting dilution. The hybridoma cells obtained by such selection are then assayed to identify clones that secrete antibodies capable of binding the polypeptide of the invention.

Alternatively, additional antibodies that can bind to a polypeptide of the invention can be generated in a two-step procedure using anti-idiotypic antibodies. Such a method
Take advantage of the fact that an antibody is itself 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 spleen cells of such animals are then used to produce hybridoma cells. The hybridoma cells are then screened to identify a clone that produces an antibody whose ability to bind to a protein-specific antibody can be blocked by a polypeptide of the invention. Such antibodies include anti-idiotypic antibodies to protein-specific antibodies, and are used to immunize animals to induce the formation of additional protein-specific antibodies.

[0981] For in vivo use of the antibodies in humans, the antibodies are "humanized." Such antibodies can be produced by using a genetic construct derived from a hybridoma cell producing the monoclonal antibody 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., U.S. Patent No. 4,816,567; Taniguchi.
Morrison et al., EP 173494; Neub.
Erger et al., WO 8615333; Robinson et al., WO 87026.
71; Boulianne et al., Nature 312: 643 (1984); N.
Euberger et al., Nature 314: 268 (1985)).

(B) Isolation of Antibody Fragments Directed to a Polypeptide from a Library of scFvs A naturally-occurring V gene isolated from human PBL can be exposed to or exposed to a donor. (See, eg, US Pat. No. 5,885,793, which is incorporated herein by reference in its entirety.) ).

[0964] Library rescue. A scFvs library is constructed from human PBL RNA as described in PCT Publication WO 92/01047. To rescue phage displaying antibody fragments, approximately 10 ⁹ E. coli harboring phagemids were rescued. E. coli is used to inoculate 50 ml of 2 × TY (containing 1% glucose and 100 μg / ml ampicillin) (2 × TY-AMP-GLU) and shake with an O.O. D. Propagate to growth. 5 of this culture
Inoculate 50 ml of 2 × TY-AMP-GLU with 2 × 10 8 TU and use 2 × 10 8 TU of Δgene 3 helper (M13Δgene III, PCT Publication WO 92/01047).
And incubate the culture without shaking at 37 ° C. for 45 minutes, then with shaking at 37 ° C. for 45 minutes. The culture is incubated at 4000 r. p. m. And resuspend the pellet in 2 liters of 2 × TY (containing 100 μg / ml ampicillin and 50 μg / ml kanamycin) and grow overnight. Phage to PCT public WO
Prepared as described in 92/01047.

[0965] 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 a greater binding avidity for the antigen. During phage morphogenesis, infectious M13Δgene III particles are generated by growing helper phage in cells carrying a pUC19 derivative that supplies the wild-type gene III protein. The culture is incubated for 1 hour at 37 ° C. without shaking, then for another hour at 37 ° C. with shaking. Centrifuge the cells (IEC-Centra 8,400r)
. p. m. / Min for 10 minutes), 100 μg / ml ampicillin and 25 μg
2 × TY broth containing 2 g / ml kanamycin (2 × TY-AMP-KAN
)) Resuspended in 300 ml and grown overnight at 37 ° C with shaking. 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) for approximately 1 μm.
A final concentration of 0 ¹³ transducing units / ml (ampicillin resistant clones) is obtained.

[0967] Panning of the library. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg / ml or 10 μg / ml of the polypeptide of the invention. Tubes were washed 3% with 2% Marvel-PBS.
Block at 7 ° C. for 2 hours, 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 turntable, and then allowed to stand for an additional 1.5 hours. The tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS.
The phage was eluted by adding 1 ml of 100 mM triethylamine and spinning up and down on a turntable for 15 minutes, after which 0.5 ml of 1.
Neutralize immediately with 0M Tris-HCl, pH 7.4. The eluted phages were then incubated with the bacteria for 30 minutes at 37 ° C., using the phages to obtain 10 ml of mid-log E. coli. E. coli TG1. Then, E. E. coli is plated on a TYE plate containing 1% glucose and 100 μg / ml ampicillin. The resulting bacterial library is then
Rescue with the Δ gene 3 helper phage as described above and prepare the phage for the next round of selection. This process was then repeated for all four times of affinity purification, with the third and fourth times washing the tubes with PBS, 0.1% Tw.
Increase by 20-fold with een-20 and 20-fold with PBS.

[0967] 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). ELISAs are 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 clones (eg, PCT publication WO
92/01047) and then further characterized by sequencing. These ELISA positive clones can also be prepared using techniques known in the art, such as epitope mapping, binding affinity, receptor signal transduction, the 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 depends on the
It requires both soluble and cognate signaling. The signal may carry a positive stimulus (allowing the cells of the B lineage to sustain programmed development) or a negative stimulus (instructing the cell to block the current generation pathway). To date, IL-2, IL-4, IL-5, IL-6, IL-7, IL
Numerous 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 B cell populations.

[0969] One of the most studied classes of B cell costimulatory proteins is the TNF superfamily. Within this family, CD40, CD27 and CD30
Together with their respective ligands, CD154, CD70 and CD153, they have been found to modulate various immune responses. Assays that allow for the detection and / or observation of the proliferation and differentiation of these B cell populations and their progenitor cells will show the effects that various proteins may 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 the differentiation, proliferation, or inhibition of B cell populations and their precursors.

[0970] In Vitro Assays-The ability of purified polypeptides of the invention, or truncated forms thereof, to induce activation, proliferation, differentiation or inhibition and / or death in B cell populations and their precursors Is evaluated. Activity of the polypeptide of the present invention on purified human tonsillar B cells (qualitatively from 0.1 to 10,000 n
(measured over a dose range of g / mL) is evaluated in a standard B lymphocyte costimulation assay. In this assay, purified tonsillar B cells were used as priming factors in formalin-fixed Staphylococcus aureus.
Culture in the presence of either Cowan I (SAC) or immobilized anti-human IgM antibody. Secondary signals, such as IL-2 and IL-15, trigger B cell proliferation in concert with SAC and IgM cross-linking as measured by tritiated thymidine incorporation. New synergists can be easily identified using this assay. This assay involves isolating human tonsillar B cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell population is CD45R
Greater than 95% B cells as assessed by expression of (B220).

[0981] Samples of each of the various dilutions were placed in individual wells of a 96-well plate, to which the culture medium (10% FBS, 5 × 10 ⁻⁵ M 2ME, 100 U / ml) was added.
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 was initiated 72 hours after addition of the factor, starting with 3H-
Quantification with thymidine (6.7 Ci / mM) with a 20 h pulse (1 μCi / well). Positive and negative controls are IL2 and medium, respectively.

In Vivo Assay-BALB / c mice are injected (ip) twice daily 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 point they were sacrificed and various tissues and sera were collected for analysis. Comparison of H & E sections from normal spleen and spleen treated with a polypeptide of the invention, results in the activity of this polypeptide in spleen cells, such as diffusion of periarterial lymphatic sheath and / or red pulp area. A significant increase in nucleated cellularity, which may indicate activation of differentiation and proliferation of the B cell population, is identified. Anti-CD, a B cell marker
Using immunohistochemical studies with 45R (B220), any physiological change to spleen cells (eg, spleen tissue disruption) causes a vaguely defined B cell compartment that infiltrates established T cell areas To determine if this is due to an increase in B cell presentation within.

Using flow cytometric analysis of spleens from mice treated with the polypeptide, the polypeptide was identified as ThB +, CD45R (B220) dull B
Shows whether the ratio of cells is specifically increased over that observed in control mice.

[0995] Furthermore, the putative consequences of the in vivo presentation of increased mature B cells is that serum I
g is a relative increase in titer. Therefore, serum IgM and IgA levels are compared between buffer-treated and polypeptide-treated mice.

The tests described in this example tested the activity of a polypeptide of the invention.
However, one of skill in the art can readily modify the illustrated studies to test the activity of the polypeptides of the invention (eg, gene therapy), agonists and / or antagonists of the polynucleotides or polypeptides of the invention. .

Example 33: T Cell Proliferation Assay A CD3-induced proliferation assay is performed on PBMC and measured by ³ H-thymidine incorporation. This assay is performed as follows. 96-well plates were loaded with 100 mAb against CD3 (HIT3a, Pharmingen).
μl / well, or isotype matched control mAb (B33.1) (
(1 μg / ml in 0.05 M bicarbonate buffer, pH 9.5) at 4 ° C. overnight,
Coat, then wash three times with PBS. PBMC were isolated from human peripheral blood by F / H gradient centrifugation and coated with mAbs in RPMI containing 10% FCS and P / S in the presence of polypeptides of the invention at various concentrations. Add to ⁴ wells (5 × 10 ⁴ / well) of plate (200 μl total).
The relevant protein buffer and medium alone are controls. After 48 hours of culture at 37 ° C., the plates are spun at 1000 rpm for 2 minutes and 100 μl
One liter of supernatant was removed and stored at −20 ° C. for measurement of IL-2 (or other cytokines) if an effect on growth was observed. 0.5 μCi in well
Supplemented with 100 .mu.l of medium containing ³ H-thymidine and at 37.degree.
Incubate for 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. A control antibody that does not induce T cell proliferation is used as a negative control for the effect of the polypeptide of the present invention.

The studies described in this example tested the activity of a polypeptide of the invention. However, one of skill in the art can readily modify the illustrated studies to test the activity of the polypeptides of the invention (eg, gene therapy), agonists and / or antagonists of the polynucleotides or polypeptides of the invention. .

Example 34: Effect of polypeptides of the invention on MHC class II, costimulatory and adhesion molecule expression, and cell differentiation of monocytes and monocyte-derived human dendritic cells Produced by the growth of proliferative progenitors found in blood: adherent PBMC or purified monocyte fractions are separated from GM-CSF (50 ng / ml) and I
Incubate with L-4 (20 ng / ml) for 7 to 10 days. These dendritic cells have the characteristic phenotype of immature cells (CD1, CD80, CD86, CD40 and M
HC class II antigen expression). Treatment with activators such as TNF-α causes rapid changes in surface phenotype (MHC class I and II, increased expression of costimulatory and adhesion molecules, down-regulation of FCγRII, up-regulation of CD83). Occurs. These changes are associated with increased antigen presenting ability and functional maturation of dendritic cells.

[0979] FACS analysis of surface antigens is performed as follows. Cells are treated with increasing concentrations of a polypeptide of the invention or LPS (positive control) for 1-3 days, 1% B
Wash with PBS containing SA and 0.02 mM sodium azide, then add 1
: Incubate with the appropriate FITC- or PE-labeled monoclonal antibody at a dilution of 20 for 30 minutes at 4 ° C. After a further wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

(Effects on Cytokine Production) Cytokines produced by dendritic cells, especially IL-12, are important in initiating T cell dependent immune responses. IL-12
Strongly influences the development of Th1 helper T cell immune responses and cytotoxic T
Induces cellular and NK cell functions. Using an ELISA, the IL
Measure -12 release. Dendritic cells (10 ⁶ / ml) are treated with increasing concentrations of the polypeptide 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 (Minneaap)
olis, MN)) to analyze for IL-12 content. Use the standard protocol provided in the kit.

[0981] Effects on MHC Class II, costimulatory and adhesion molecule expression. Three main families of cell surface antigens: adhesion molecules, molecules involved in antigen presentation, and Fc receptors can be identified on monocytes. Modulation of the expression of MHC class II antigens and other costimulatory molecules (eg, B7 and ICAM-1) can alter the ability of monocytes to present antigen and induce T cell activation. Increased expression of the Fc receptor may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.

[0981] FACS analysis is used to test for surface antigens as follows. Monocytes were incubated with increasing concentrations of a polypeptide of the invention or LPS (positive control) at 1
PB containing 1% BSA and 0.02 mM sodium azide for ~ 5 days
Wash with S, then incubate with the appropriate FITC- or PE-labeled monoclonal antibody at a 1:20 dilution for 30 minutes at 4 ° C.
After further washing, the labeled cells were washed with FACScan (Becton Dickin).
son) and analyzed by flow cytometry.

[0983] Molecules that activate (or inactivate) monocytes and / or increase monocyte survival (or reduce monocyte survival) Assays are known in the art and can be routinely applied to determine whether a molecule of the invention functions as a monocyte inhibitor or activator. A polypeptide, agonist or antagonist of the present invention can be screened using the three assays described below. For each of these assays, peripheral blood mononuclear cells (PBMCs) were
A single donor leuko was obtained by centrifugation through an aque gradient (Sigma).
pack (American Red Cross, Baltimore, MD
). Monocytes are counter-currently centrifuged elutriated (counterflow).
isolated from PBMC by w centrifugal elutriation.

[0984] Monocyte Survival Assay. Human peripheral blood monocytes gradually lose viability when cultured in the absence of serum or other stimuli. Their death results from an internally regulated process (apoptosis). Addition of activators, such as TNFα, to the culture dramatically improves cell survival and prevents DNA fragmentation. Propidium iodine (
Apoptosis is measured using PI) staining as follows. Monocytes, 100n
Culture 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 concentration of 2 × 10 ⁶ / ml in PBS containing PI at a final concentration of 5 μg / ml, and then incubated for 5 minutes at room temperature before FACScan analysis. PI incorporation 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 in the absence of this polypeptide under the same conditions. For production of IL-12, the cells are primed overnight with IFN (100 U / ml) in the presence of a polypeptide of the invention. Next, LPS (10
ng / ml). Conditioned media is collected after 24 hours and stored frozen until use. The measurement of TNF-α, IL-10, MCP-1 and IL-8 was then performed using a commercially available ELISA kit (eg, R & D Systems Minne).
apolis, MN) and applying the standard protocols provided in the kit.

(Oxidative burst) Purified monocytes were collected from 96
Plate well plates at 2-1 × 10 ⁵ cells / well. Increasing concentrations of the polypeptide of the invention were added to a total well volume of 0.2 ml of 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. In a monolayer of macrophages, 0.2 ml of a 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). Plate at 37 ° C for 2
Incubate for hours and stop the reaction by adding 20 μl per well of 1 N NaOH. Read the absorbance at 610 nm. 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 a polypeptide of the invention. However, those skilled in the art will recognize that the activity of the polypeptide of the present invention, the activity of the polynucleotide (
The exemplified studies can be readily modified to test the activity of, for example, gene therapy, agonists, and / or antagonists.

Example 35 Biological Effects of the Polypeptides of the Invention Astrocytes and Neuronal Assays As described above, recombinant recombinant polypeptides of the invention expressed in Escherichia coli and purified Can be tested for activity in promoting cortical neuronal cell survival, neurite outgrowth or phenotypic differentiation, and for inducing proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. Selection of cortical cells for bioassays is based on the widespread expression of FGF-1 and FGF-2 in cortical structures and 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.

[0987] Furthermore, FGFs to cortical or hippocampal neurons in vitro
Previous reports describing the biological effects of -2 (basic FGF) 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 neuroite 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 not only which FGF is being tested, but which receptor is expressed in the target cell. Suggests that you 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, for example using a thymidine incorporation assay.

[0987] Fibroblast and Endothelial Cell Assays Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.) And maintained in growth media from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, CA). For proliferation assays, human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells / well in growth media in 96-well plates for 1 day. The cells are then incubated for one 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) to each well to a final concentration of 10%. The cells are incubated for 4 hours. Cell viability
Measured by reading on a ytoFluor fluorescence reader. For PGE ₂ assays, the human lung fibroblasts, one day in a 96-well plate, cultured at 5,000 cells / well. After changing the medium to 0.1% BSA basal medium, the cells were
Incubate with FGF-2 or a polypeptide of the invention with or without L-1α for 24 hours. 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 in a 96-well plate for 1 day. After changing the medium to 0.1% BSA basal medium, the cells are incubated with FGF-2 with or without the polypeptide IL-1α of the present invention for 24 hours. Supernatants were collected and ILs were collected using an ELISA kit (Endogen, Cambridge, MA).
Assay for -6.

Human lung fibroblasts were cultured with FGF-2 or a polypeptide of the present invention in basal medium for 3 days, and then Alam to assess the effect on fibroblast proliferation.
Add ar Blue. FGF-2 should exhibit a stimulus of 10 to 2500 ng / ml that can be used comparable to stimulation with a polypeptide of the invention.

Parkinson's Model Loss of motor function in Parkinson's disease is due to striatal dopamine deficiency resulting from degeneration of dopaminergic projection neurons in the substantia nigra striatal. An animal model of Parkinson's disease that has been extensively characterized is 1-methyl-4phenyl 1,2,3
, 6-tetrahydropyridine (MPTP). In the CNS,
MPTP is taken up by astrocytes and is mediated by monoamine oxidase B
-Catalyzed to -methyl-4-phenylpyridine (MPP ⁺ ) and released.
Subsequently, MPP ⁺ actively accumulates in dopaminergic neurons via the high-affinity reuptake transporter of dopamine. MPP ⁺ is then enriched in mitochondria by an electrochemical gradient and selectively inhibits nicotinamide diphosphate: ubiquinone oxidoreductase (complex I), thereby disrupting electron transfer and Generates active oxygen.

It has been demonstrated in a tissue culture paradigm that FGF-2 (basic FGF) has trophic activity on substantia nigra dopaminergic neurons (Ferrari)
Dev. Biol. 1989). In recent years, Dr. Unsicker's group has demonstrated that administration of FGF-2 in a striatum gel-type implant results in nearly complete protection of substantia nigra dopaminergic neurons from the toxicity associated with MPTP exposure ( Otto and Unsicker, J. Neuroscience,
1990).

[0995] Based on data using FGF-2, the polypeptides of the present invention have similar effects to those of FGF-2 in enhancing dopaminergic neuron survival in vitro. Polypeptides of the invention can also be evaluated in vivo to determine whether they have, and dopaminergic neurons in the striatum can be tested 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 neuron cell culture paradigm. 14 days of pregnancy
Cultures are prepared by dissecting the midbrain plate from r-rat embryos. Tissues were dissociated with trypsin and seeded at a density of 200,000 cells / cm ^{2 on} polyortinin-laminin coated coverslips. The cells are maintained in Dulbecco's modified Eagle's medium and F12 medium containing hormone supplement (NI). After 8 days in vitro, the 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 three days, and the factor is also added every time.

Since dopaminergic neurons are isolated from animals at day 14 of gestation (the time of development is past the stage where dopaminergic progenitor cells proliferate), the increase in the number of tyrosine hydroxylase immunopositive neurons is 2 shows an increase in the number of surviving dopaminergic neurons. Thus, if a polypeptide of the present invention acts to prolong dopaminergic survival, it indicates that the polypeptide may be involved in Parkinson's disease.

The studies described in this example tested the activity of a polypeptide of the invention. However, those skilled in the art will appreciate the activity of the polynucleotides of the invention (eg, gene therapy).
The illustrated studies can be readily modified to test agonists and / or antagonists of the 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 treated with 4% fetal bovine serum (FBS).
), 16 units / ml heparin, and 50 units / ml endothelial cell growth supplement (ECGS, Biotechnique, Inc.).
Seed in medium at a density of 2-5 x 10 ⁴ cells per 35 mm dish. On day 2, the medium was washed with 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 days 4 and 6, the medium is changed. Day 8, Coulter C
The number of cells is determined using a counter.

[0998] An increase in the number of HUVEC cells indicates that the polypeptides of the invention are capable of proliferating vascular endothelial cells.

The studies described in this example tested the activity of a polypeptide of the invention. However, those skilled in the art will appreciate the activity of the polynucleotides of the invention (eg, gene therapy).
The illustrated studies can be readily modified to test agonists and / or antagonists of the invention.

(Example 37: Stimulating effect of the polypeptide of the present invention on the proliferation of vascular endothelial cells) [1000] Colorimetric analysis using the electron conjugate reagent PMS (phenazine methosulfate) for evaluating the mitogenic activity of growth factors An analytical MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -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 medium and allowed to attach overnight. 0
. After 12 hours serum starvation in 5% FBS, conditions (bFGF, VEGF ₁₆₅ or polypeptide of the invention in 0.5% FBS) with or without Heparin (8 U / ml) were added to the wells for 48 hours Added. 20mg MTS / PMS
The mixture (1: 0.05) is added per well and allowed to incubate at 37 ° C. for 1 hour, after which the absorbance at 490 nm is measured on an ELISA plate reader. The background absorbance of control wells (with media, no cells) is subtracted and 7 wells are run in parallel for each condition.
Leak et al., In Vitro Cell. Dev. Biol. 30A: 512
518 (1994).

[1001] The studies described in this example tested the activity of a polypeptide of the invention. However, those skilled in the art will appreciate the activity of the polynucleotides of the invention (eg, gene therapy).
The illustrated studies can be readily modified to test agonists and / or antagonists of the invention.

Example 38 Inhibition of PDGF-Induced Vascular Smooth Muscle Cell Growth Stimulating Effect HAoSMC proliferation can be measured, for example, by BrdUrd incorporation. Briefly, subconfluent stationary phase cells growing on 4-chamber slides were converted to 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 the 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 a denaturing solution, and then with streptavidin-peroxidase and diaminobenzidine. After counterstaining with hematoxylin, the cells are mounted for microscopic examination and BrdUrd-positive cells are counted. BrdU
The rd coefficient is calculated as the ratio of BrdUrd-positive cells to the total cell number. In addition, simultaneous detection of BrdUrd staining (nuclei) and FITC incorporation (cytoplasm) is performed for individual cells by the simultaneous use of light and dark UV fluorescent illumination. (Hayashida et al., J. Biol. Chem. 6: 271 (3
6): 21985-19992 (1996)).

The studies described in this example tested the activity of a polypeptide of the invention.
However, those skilled in the art will appreciate that the activity of the polynucleotides of the invention (eg, gene therapy),
The illustrated studies can be readily modified to test agonists and / or antagonists of the invention.

Example 39: Stimulation of Endothelial Migration This example can be used to explore the potential of polypeptides of the invention to stimulate lymphatic endothelial cell migration.

The endothelial cell migration assay is performed using a 48-well microchemotaxis chamber (Neuroprobe Inc., Cabin John, MD; Falk).
J., W et al. Immunological Methods 1980; 33:
239-247). Polycarbonate filter without polyvinylpyrrolidone, which has a pore size of 8 μm (Nucleopore Corp. Cam
bridge, MA) at room temperature for at least 6 hours with 0.1% gelatin,
Then dry under sterile air. The test substance is 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 apparatus. Wash sub-confluent, early passage (2-6) HUVEC or BMEC cultures and trypsinize for the minimum time necessary for detachment of cells. After placing the filter between the bottom and top chambers, the upper compartment is seeded with 2.5 × 10 ⁵ cells suspended in 50 μl M199 containing 1% FBS. This device is then
Cells were allowed to migrate by incubation at 37 ° C. for 5 hours in a chamber humidified with 5% CO 2. After the incubation period, the filter was removed and the top side of the filter with non-migrating cells was replaced with a rubbery policeman (polic).
eman). The filter was fixed with methanol and Giem
Stain with Sa solution (Diff-Quick, Baxter, McGraw P
ark, IL). Migration is quantified by counting cells in three random high power fields (40 ×) in each well. Then, all groups are executed in quadruplicate.

The studies described in this example tested the activity of a polypeptide of the invention.
However, those skilled in the art will appreciate that the activity of the polynucleotides of the invention (eg, gene therapy),
The illustrated studies can be readily modified to test agonists and / or antagonists of the invention.

Example 40: Stimulation of Nitric Oxide Production by Endothelial Cells Nitric oxide released by the vascular endothelium is thought to be a mediator of vascular endothelial relaxation. Accordingly, the activity of a polypeptide of the invention can be assayed by measuring nitric oxide production by endothelial cells in response to the polypeptide.

[1010] Nitric oxide was starved for 24 hours, followed by various levels of positive control (
For example, confluent microvascular endothelial cells are measured in 96-well plates after 4 hours of exposure to VEGF-1) and a polypeptide of the invention. Nitric oxide in the medium is measured by using the Griess reagent to determine the total amount of nitrous acid after reduction of nitric acid derived from nitric oxide by nitrate reductase. The effect of the polypeptides of the invention on nitric oxide release is tested in HUVEC.

[1010] Briefly, NO release from cultured HUVEC monolayers was measured using a NO meter (Iso
-NO, World Precision Instruments Inc.
) Measure using a NO-specific polarographic electrode connected to (1049).
Calibration of the NO element is performed according to the following equation: 2KNO ₂ + 2KI + 2H ₂ SO ₄ 62NO + I ₂ + 2H ₂ O + 2K ₂ SO _{4 A} calibration curve is prepared in a calibration solution containing KI and H ₂ SO ₄ at graded concentrations of KNO ₂ (
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 HUVEC is washed twice with Dulbecco's phosphate buffered saline. The cells were then 5m in a 6 well plate.
of filtered Krebs-Henseleit solution and slide the cell plate on a slide warmer (Lab Lin) to maintain the temperature at 37 ° C.
e Instruments Inc. ). The 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 picomoles per 1 × 10 ⁶ endothelial cells. All reported values are the 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).

[1010] The studies described in this example tested the activity of a polypeptide of the invention.
However, one of skill in the art can readily modify the illustrated studies to test the activity of a polynucleotide (eg, gene therapy), agonist, and / or antagonist of the present invention.

Example 41: Effect of the polypeptides of the invention on cord formation in angiogenesis Another step in angiogenesis is in the cord (c) characterized by endothelial cell differentiation.
ord) formation. This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in vitro.

[1012] CADEM (microvascular endothelial cells) was used as a proliferating cell (passage 2) as Cell
Applications Inc. Purchased from Cell Applica
cultivated in IONs' CADME Growth Medium, and
Use at the passage. For the in vitro angiogenesis assay, the wells of a 48-well cell culture plate were prepared using Cell Applications' Attachme.
Coat using nt Factor Medium (200 ml / well) at 37 ° C. for 30 minutes. CADME was added to the coated wells at 7,500 cells /
Seed in wells and culture overnight in Growth Medium. The Growth Medium was then added to 300 mg of Cell Applica containing control buffer or polypeptide of the invention (0.1-100 ng / ml).
Replace with Tions' Chord Formation Medium and incubate the cells for an additional 48 hours. The number and length of capillary-like cords were
Quantification is through the use of an eler VIA-170 video image analyzer. All assays are performed in triplicate.

[1013] Commercially available (R & D) VEGF (50 ng / ml) is used as a positive control. β-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 a polypeptide of the invention. However, one of skill in the art can readily modify the illustrated studies to test the activity of a polynucleotide (eg, gene therapy), agonist, and / or antagonist of the present invention.

Example 42: Angiogenic Effect on Chick Chorioallantoic Membrane Chick 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 CAMs can be tested.

[1016] Fertilized eggs of White Leghorn chickens (Gallus gallus) and Japanese quail (Coturnix coturnix)
Incubate at 37.8 ° C and 80% humidity. 16 day old chicken differentiated CAM and 13 day old quail embryos are studied in the following manner.

At day 4 of development, a window is made in the eggshell of the chicken egg. The embryo is examined for normal development, and the eggs are sealed with Cellotape®. Add one more
Incubate until day 3. Thermanox cover glass (Nunc,
Naperville, IL) into disks about 5 mm in diameter. The sterile and salt-free growth factor is dissolved in distilled water and about 3.3 mg / 5 ml is pipetted to a disk. After air drying, apply the inverted disc to the CAM. Three days later, three specimens
% Glutaraldehyde and 2% formaldehyde and 0.12
Rinse with M sodium cacodylate buffer. These were converted to a stereoscopic microscope [Wild M8
] And embedded for semi-thin and ultrathin sectioning as described above. Control is performed only with the carrier disk.

The studies described in this example tested the activity of a polypeptide of the invention.
However, one of skill in the art can readily modify the illustrated studies to test the activity of a polynucleotide (eg, gene therapy), agonist, and / or antagonist of the present invention.

Example 43: Angiogenesis Assay Using Matrigel Implants in Mice [1010] The in vivo angiogenesis assay of the polypeptides of the present invention is based on the pre-existing capillary network-like structure of a mouse extracellular matrix material (Matrigel). 2.) Measure the ability to form new vessels in the implanted capsule. This protein is converted to liquid Ma at 4 ° C.
Mix with trigel and then inject the mixture subcutaneously into mice, where the mixture solidifies. After 7 days, the Matrigel solid "plug" is removed and tested for the presence of new blood vessels. Matrigel to Bect
on Dickinson Labware / Collaborative B
Purchased from iomedical Products.

[1020] When thawed at 4 ° C, the Matrigel material is a liquid. This Matrige
1 is mixed with the polypeptide of the present invention at 150 ng / ml at 4 ° C. and placed in a cold 3 ml syringe. Female C57BI / 6 mice, approximately 8 weeks of age, are injected with a mixture of Matrigel and experimental protein at two sites on the mid-ventral surface of the abdomen (0.
5 ml / site). Seven days later, the mice were sacrificed by cervical dislocation and Matrige
Remove the plug and wash (ie, remove any attached membranes and fibrous tissue). The entire duplicate plug is fixed in neutral buffered 10% formamide, embedded in paraffin, and used to make sections for histological examination after staining with Masson's Trichrome. Sections from three different areas of each plug are processed. Selected sections are stained for the presence of vWF. The positive control for this assay was 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 activity of a polypeptide of the invention.
However, one of skill in the art can readily modify the illustrated studies to test the activity of a polynucleotide (eg, gene therapy), agonist, and / or antagonist of the present invention.

Example 44: Rescue of ischemia in a rabbit lower limb model To study the in vivo effects of polynucleotides and polypeptides of the invention on ischemia, a rabbit hind limb ischemia model was described previously. (Takesh
ita et al., Am J. et al. Pathol 147: 1649-1660 (1995)
1) by surgical removal of one femoral artery. Resection of the femoral artery results in retrograde propagation of thrombus and occlusion of the external iliac artery. As a result, blood flow to the ischemic limb is dependent on collateral vessels arising from the internal iliac artery (Takeshita et al., Am J
. Pathol 147: 1649-1660 (1995)). At 10-day intervals, allow post-operative recovery of rabbits and development of endogenous collateral vessels. After surgery 1
On day 0 (day 0), after performing a baseline angiography, the internal iliac artery of the ischemic limb was excised using 500 mg of the naked expression plasmid containing the polynucleotide of the present invention.
(Riessen et al., Hum Gene Ther. 4: 749-758 (19
93); Leclerc et al. Clin. Invest. 90: 936-94
4 (1992)) by the arterial gene transfer technique using a hydrogel-coated balloon catheter. When a polypeptide of the invention is used for treatment, a single bolus of 500 mg of a polypeptide of the invention or control is delivered via an infusion catheter over 1 minute into the internal iliac artery of the ischemic limb. On day 30, various parameters are measured in these rabbits: (a) ratio of BP ratio-systolic blood pressure of ischemic limb to systolic blood pressure of normal limb; (b) blood flow and reflux-stop FL. : Blood flow during the non-dilated state, and max FL: blood flow during the fully dilated state (also an indirect measurement of vascular volume), and reflux is max FL: stop F
(C) angiographic score
e)-This is determined by angiography of collateral vessels. Values are determined by percentage of circles in the overlaid grid (using transversely opaque arteries divided by the total number of rabbit thighs m); (d) capillary density-determined on light microscopy sections from hind limbs. Number of collateral capillaries.

The studies described in this example tested the activity of the polynucleotides and polypeptides of the present invention. However, one of ordinary skill in the art can readily modify the illustrated studies,
An agonist and / or antagonist of the invention may be tested.

Example 45: Effect of the polypeptide of the present invention on vasodilation Since dilation of vascular endothelium is important for lowering blood pressure, spontaneously hypertensive rats (SHR)
In), the ability of the polynucleotide of the invention to affect blood pressure is tested. Increasing doses (0, 10, 30, 100, 300, and 900 mg / kg) of a polypeptide 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 for response to buffer alone.

[1025] The studies described in this example tested the activity of a polypeptide of the invention.
However, one of skill in the art can readily modify the illustrated studies to test the activity of a polynucleotide (eg, gene therapy), agonist, and / or antagonist of the present invention.

Example 46: Rat Ischemic Skin Flap Model Evaluation parameters include skin blood flow, skin temperature, and factor VIII immunohistochemistry or endothelial alkaline phosphatase reaction. The expression of the polypeptides of the invention during cutaneous 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 the following: a) A 3 × 4 cm single stem full thickness random skin flap (single pedicle)
creating a full-thickness random skin flap (muscular flap extending to the lower back of the animal); b) making a resected wound (4-6 mm in diameter) on ischemic skin (flap); c) 1-100 mg Topical treatment of a resected wound (at 0, 1, 2, 3, 4 days after wounding) with a polypeptide of the invention in various dosage ranges of: d) histological study For immunohistochemical and in situ studies,
Harvest wound tissue on days 3, 5, 7, 10, 14, and 21 after wounding.

The study described in this example tested the activity of a polypeptide of the invention.
However, one of skill in the art can readily modify the illustrated studies to test the activity of a polynucleotide (eg, gene therapy), agonist, and / or antagonist of the present invention.

Example 47: Peripheral Artery Disease Model Angiogenesis Therapy Using the Polypeptides of the Invention is a Novel Therapeutic Strategy for Restoring Peri-ischemic Blood Flow in Peripheral Artery Disease . The experimental protocol includes: a) The femoral artery on one side is ligated to create the ischemic muscle of the hind limb, the hind limb on the other side serving as a control.

B) Deliver the polypeptide of the present invention in a dosage range of 20 mg to 500 mg, 3 times (possibly more) intravenously and / or intramuscularly per week 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 in the normal muscle of the contralateral hind limb on the other side.

The studies described in this example tested the activity of a polypeptide of the invention. However, one of skill in the art can readily modify the illustrated studies to test the activity of a polynucleotide (eg, gene therapy), agonist, and / or antagonist of the present invention.

Example 48: Ischemic Cardiomyopathy Model The polypeptides of the present invention are evaluated as potent mitogens that can stimulate collateral vessel development and reconstitute new blood vessels after coronary artery occlusion. . Changes in the expression of the polypeptide are investigated in situ. The experimental protocol includes: a) The heart is exposed via a left thoracotomy of the rat. Immediately, the left coronary artery
Occlude with a thin suture (6-0) and close the rib cage.

B) The polypeptide of the invention is delivered in a dosage range of 20 mg to 500 mg three times (possibly more) intravenously and / or intramuscularly per week for 2 to 4 weeks.

C) Thirty days after surgery, the heart is removed and sectioned for morphometry and analyzed in situ.

The studies described in this example tested the activity of a polypeptide of the invention.
However, one of skill in the art can readily modify the illustrated studies to test the activity of a polynucleotide (eg, gene therapy), agonist, and / or antagonist of the present 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) Making an incision 1-1.5 mm in length from the center of the cornea to the stromal layer.

B) Inserting a spatula under the lip margin of the incision facing the outer corner of the eye.

C) Creating a pocket (the base of which is 1-1.5 m from the edge of the eye)
m).

D) Placing a pill containing 50 ng to 5 μg of a polypeptide of the invention in a pocket.

E) Treatment with a polypeptide of the invention may also be applied topically to corneal wounds within a dosage range ranging from 20 mg to 500 mg (daily treatment for 5 days).

The studies described in this example tested the activity of a polypeptide of the invention. However, one of skill in the art can readily modify the illustrated studies to test the activity of a polynucleotide (eg, gene therapy), agonist, and / or antagonist of the present invention.

Example 50 Diabetic Mice and Glucocorticoid-Injured Wound Healing Model A. Diabetic db + / db + Mouse Model To demonstrate that the polypeptides of the invention enhance the healing process, A genetic diabetes mouse model is used. The full thickness wound healing model in db + / db + mice is a well-characterized, clinically relevant and reproducible model of wound wound healing. Healing of diabetic wounds relies on granulation tissue formation and re-epithelialization rather than contraction (Gartner, MH, et al., J. Surg. Res. 52: 389 (19).
92); Greenhalgh, D .; G. FIG. Et al., Am. J. Pathol. 136
: 1235 (1990)).

This diabetic animal has many of the characteristic features observed in type II diabetes mellitus. Homozygous (db + / db +) mice have their normal heterozygous (
db + / + m) Obese compared to littermates. Mutant diabetes (db + / db +)
The mouse has a single autosomal recessive mutation (db +) on chromosome 4 (C
Oleman et al., Proc. Natl. Acad. Sci. USA 77:28
3-293 (1982)). Animals exhibit polyphagia, polydipsia, 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 .; Et al., Clin. Exp. Immunol. 51 (1): 1
-7 (1983); Leiter et al., Am. J. 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 (
Norido, F .; Et al., Exp. Neurol. 83 (2): 221-232 (
1984); Robertson et al., Diabetes 29 (1): 60-6.
7 (1980); Giacomelli et al., Lab Invest. 40 (4)
Coleman, D .: 460-473 (1979); L. , Diabetes
31 (supplement): 1-6 (1982)). These homozygous diabetic mice develop hyperglycemia, which is similar to human type II diabetes and is resistant to insulin (Mandel et al., J. Immunol. 120: 1375-13).
77 (1978)).

The characteristics observed in these animals indicate that healing in this model may be similar to the healing observed in human diabetes (Greenhalgh et al., Am. J. of Pathol. 136: 1235-1246 ( 1990))
.

[1045] Genetically diabetic female C57BL / KsJ (db + / db +) mice and their non-diabetic (db + / + m) heterozygous littermates are used in this study (J
ackson Laboratories). These animals were purchased at the age of 6 weeks,
And these animals are 8 weeks old at the start of the study. Animals are housed individually and are given food and water ad libitum. All operations are performed using aseptic techniques. This experiment was performed using the Human Genome Sciences, Inc.
Tional Animal Care and Use Committee
and the Guidelines for the Care and
This is performed according to the rules and guidelines of Use of Laboratory Animals.

[1045] The wound protocol is based on 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, animals were treated with A dissolved in deionized water.
Anesthetize with intraperitoneal injection of vertin (0.01 mg / mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol. The dorsal area of the animal is shaved and the skin is washed with a 70% ethanol solution and iodine. The surgical area is dried with sterile gauze before wounding. The 8 mm full-thickness wound is then
Prepared using a Keyes tissue punch. Immediately after wounding, the surrounding skin is gently stretched to remove wound enlargement. The wound is allowed to open during the experiment. Application of treatment is given topically for 5 consecutive days starting from the day of wounding. Prior to treatment, the wound is gently washed with sterile saline and gauze sponge.

The wounds are visually inspected and photographed at a fixed distance on the day of surgery and two days thereafter. Wound closure is determined by daily measurements on days 1-5 and day 8. Wounds are 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 continuous epithelium.

The polypeptides of the present invention are administered in a 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.

The animals are euthanized on day 8 with an 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 a tissue cassette between biopsy sponges for further processing.

[1010] 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.

[1045] Wound closure is analyzed by measuring the area on the horizontal and vertical axes and obtaining the sum of the areas of the wound. Contraction is then assessed by establishing the difference between the area of the original wound (day 0) and the area after treatment (day 8). The wound area on day 1 is 64 mm ² (size corresponding to a 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] Samples are fixed in 10% buffered formalin and paraffin The 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 transverse sections of bisected wounds. Histological examination of the wound is used to assess whether the healing process and morphological appearance of the repaired skin has been altered by treatment with a polypeptide of the invention. This assessment includes verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, reepithelialization, and epidermal maturation (Gree
nhalgh, D.M. G. FIG. Et al., Am. J. Pathol. 136: 1235 (19
90)). Using a graduated lens micrometer with a blinded observer
observer).

The tissue sections are also immunohistochemically stained with a polyclonal rabbit anti-human keratin antibody using the ABC Elite detection system. Human skin is used as a positive tissue control, while non-immune IgG is used as a negative control.
Keratinocyte proliferation is determined by assessing the extent of wound re-epithelialization using a graduated lens micrometer.

Proliferating cell nuclear antigen / cyclin (PCNA) in skin specimens was assayed for ABC El
Demonstrated by using anti-PCNA antibody (1:50) with an item 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 contains sections with primary antibody shedding and replacement with non-immune mouse IgG. The ranking of these sections is based on the degree of growth on a scale of 0-8 (lower scale reflecting slight growth to higher reflecting strong growth).

[1056] The experimental data is analyzed using a one-tailed t-test. A p-value of <0.05 is considered significant.

B. Steroid Impaired Rat Model Inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids and
d Wound hearing: Anti-Inflammatory St
eroid Action: Basic and Clinical Aspe
cts. 280-302 (1989); Wahl et al. Immunol. 1
15: 476-481 (1975); Werb et al. Exp. Med. 147
1684-1694 (1978)). Glucocorticoids inhibit angiogenesis, vascular permeability (Ebert et al., An. Intern. Med. 37: 7).
01-705 (1952)), fibroblast proliferation, and collagen synthesis (Bec
k et al., Gross Factors. 5: 295-304 (1991); Hay.
nes et al. Clin. Invest. 61: 703-797 (1978)).
As well as causing a transient decrease in circulating monocytes (Hay
nes et al. Clin. Invest. 61: 703-797 (1978);
Wahl, "Glucocorticoids and wound seal.
ing ": Antiinflammation Steroid Action
: Basic and Clinical Aspects, Academic
Press, New York, 280-302 (1989)). Systemic administration of steroids to impaired wound healing is a well-established phenomenon in rats (Beck et al., Growth Fact).
ors. 5: 295-304 (1991); Haynes et al. Clin.
Invest. 61: 703-797 (1978); Wahl, "Gluco.
corticoids and wound hearing ": Antiin
Flammatory Steroid Action: Basic and
Clinical Aspects, Academic Press, New
York, 280-302 (1989); Pierce et al., Proc. Nat
l. Acad. Sci. ScL USA 86: 2229-2233 (1989)).

To demonstrate that the polypeptides of the present invention can enhance the healing process, a plurality of polypeptides of the present invention on rat full thickness skin cut wounds where healing is impaired by systemic administration of methylprednisolone To evaluate the effect of topical application of.

[1059] Young adult male Sprague Dawley rats weighing 250-300 g
) (Charles River Laboratories) is used in this experiment. The animals are purchased at the age of 8 weeks. At the start of the study, he is 9 weeks old. The healing response of rats was determined at the time of wounding by systemic administration of methylprednisolone (17 mg / k
g / rat, intramuscular). Animals are housed individually and are given food and water ad libitum. All operations are performed using aseptic techniques. Huma
n Institutional of Genome Sciences, Inc
Animal Care and Use Committee and th
e Guidelines for the Care and Use of
Perform according to Laboratory Animal Rules and guidelines.

The wound protocol follows section A above. On the day of wounding, the animals are treated with ketamine (5 m
g / kg) and xylazine (5 mg / kg) by intramuscular injection. The dorsal area of the animal is shaved and the skin is washed with a 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, open the left side of the wound. Application of the test substance is given topically once daily 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 wounds are 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 measurements on day 8. Wounds are 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 continuous epithelium.

The polypeptides of the present invention are administered in a 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 the vehicle solution.

The animals are euthanized on day 8 with an 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 a tissue cassette between biopsy sponges for further processing.

[1064] Four groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) untreated group, 2) vehicle placebo control, and 3) treated group. .

The wound closure is analyzed by measuring the area on the vertical and horizontal axes and obtaining the sum of the wound areas. Closure is then assessed by establishing the difference between the area of the original wound (day 0) and the area after treatment (day 8). The wound area on day 1 is 64 mm ² (size corresponding to a 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] Samples are fixed in 10% buffered formalin and paraffin The embedded mass is cut perpendicular to the wound surface (5 mm) and cut with an Olympus microtome. Routine hematoxylin-eosin (H & E) staining is performed on transverse sections of bisected wounds. Histological examination of the wound makes it possible to assess whether the healing process and the morphological appearance of the repaired skin have been improved by treatment with the polypeptides according to the invention. A graduated lens micrometer is used by a blinded observer to determine the distance of the wound gap.

[1067] The experimental data is analyzed using a one-tailed t-test. A p-value of <0.05 is considered significant.

The studies described in this example tested the activity of a polynucleotide of the invention. However, one of skill in the art can readily modify the illustrated studies to test the activity of the polypeptides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 51: Lymphadema Animal Model The purpose of this experimental approach was to establish lymphatic vessel formation (lymphh) in rat hind limbs.
a suitable and consistent model of lymphedema to test the therapeutic effect of the polypeptides of the present invention in the re-establishment of the lymphatic circulatory system. Efficacy is measured by swelling volume of the affected limb, quantification of the amount of lymphatic vessels, amount of 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.

[1010] Before beginning surgery, blood samples are drawn for protein concentration analysis.
Male rats (weighing about 350 g) are dosed with pentobarbital. Next, the right limb is 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 test. After measuring the circumference and volume, two measurement levels (0.5 cm above the heel, midpoint of the back of the foot) are marked, and the foot is injected with dye. On both right and left dorsal endothelium,
Inject 0.05 ml of 1% Evan's Blue. Circumferential and volume measurements are then made after injection of the dye into the paw.

[1070] Using the knee joint as a landmark, a mid-limb inguinal incision is made annularly so that the location of the femoral vessels can be confirmed. The flap is dissected and separated using forceps and hemostat. After locating the femoral vessels, locate the lymph vessels that run alongside and below the vessels. The major lymphatic vessels in this area were then ligated with an electrocoagulated suture ligature (elevated).
critically ligate).

Using a microscope, the muscles in the back of the limb (semitendinosis)
And near the adductor muscle). The location of the popliteal lymph nodes 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 accompanying fat tissue are then removed by cutting connective tissue.

Be careful to manage any mild bleeding that occurs in this procedure. After occlusion of the lymphatic vessels, the skin flap was replaced with liquid skin (Vetbond) (AJ Buck).
Seal by using. The separate skin margins are sealed to the underlying muscle tissue, leaving a gap of about 0.5 cm around the leg. The skin may also be anchored when necessary by suturing to the underlying muscle.

The animals are individually housed using mesh (no bedding) to avoid infection. The recovered animals are checked daily through the optimal edema peak. This peak typically occurs for up to 5-7 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 is determined and it is also investigated whether protein analysis is a useful test perimeter. The weights of both the control limb and the edema limb are evaluated in two places. The analysis is performed in a blind manner.

[1078] Circumference measurement: Under short-term gas anesthesia to prevent limb movement, measure the limb circumference 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 obtained from both the control and edema paw.

[1075] Volume measurement: On the day of surgery, animals are anesthetized with pentobarbital and tested prior to surgery. For daily volume measurements, the animals are placed under short-term halothane anesthesia (rapid fixation and quick recovery), both legs are shaved and the legs are marked equally with a water-resistant marker. The legs are first dipped in water, then dipped into the device to each marked level, and then the Buxco edema software (Chen / Vict).
or). The data is recorded by one person, while the other dips into the marked area.

Blood-Plasma Protein Measurement: Blood is removed, centrifuged, and serum separated before surgery, and then concluded for total protein and Ca2 + comparison.

[1078] Limb Weight Comparison: After blood is drawn, the animals are prepared for tissue collection.
The limb is cut with quiltine, then both the experimental and control legs are ligated and cut and weighed. A second weighing is performed by removing the tibio-cacaneal joint and weighing the foot.

[1078] Histological preparation: Transverse muscle located behind the knee (popliteal) area is excised and placed in a metal mold, filled with freezeGel, immersed in cold methyl butane, at -80EC until cut into labeled sample bags. To place. At the time of the cut, the muscle is observed for lymphatic vessels under a fluorescent microscope.

The studies described in this example tested the activity of a polypeptide of the invention.
However, one of skill in the art can readily modify the illustrated studies to test the activity of a polynucleotide (eg, gene therapy), agonist, and / or antagonist of the present invention.

Example 52 Inhibition of TNFα-Induced Adhesion Molecule Expression by the Polypeptides of the Invention [1980] Recruitment of lymphocytes to areas of inflammation and angiogenesis is associated with cell surface adhesion molecules (CAMs) on lymphocytes. It concerns specific receptor-ligand interactions with the vascular endothelium. This adhesion process involves both intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-
1), followed by a multi-step cascade involving endothelial leukocyte adhesion molecule-1 (E-selectin) expression in endothelial cells (EC). The expression of these and other molecules on the vascular endothelium determines the efficiency with which leukocytes can adhere to the local vasculature and extravasate to local tissues 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), a potent pro-inflammatory cytokine, is a stimulator of all three CAMs in endothelial cells and can be involved in a wide variety of inflammatory responses, often Produces a scientific result.

The potential of the polypeptides of the invention to mediate the suppression of TNF-α-induced CAM expression can be tested. The amount of CAM expression in TNF-α treated protein EC when co-stimulated with a member of the FGF family of proteins using a modified ELISA assay, which uses EC as a solid phase sorbent, was determined. Measure.

To perform this experiment, human umbilical vein endothelial cell (HUVEC) cultures were obtained from pooled cord harvest and grown in growth medium (EGM-2; supplemented with 10% FCS and 1% penicillin / streptomycin). Clonetics, San Die
(go, CA) in a humidified incubator at 37 ° C. with 5% CO ₂ . HUVECs are seeded at a concentration of 1 × 10 ⁴ cells / well in EGM medium in 96-well plates at 37 ° C. for 18-24 hours or until confluent. Subsequently, the monolayer is washed three times with a serum-free solution of RPMI-1640 supplemented with 100 U / ml penicillin and 100 mg / ml streptomycin, and at 37 ° C. for 24 hours with the given cytokines and / or growth factors. Processed. After incubation, the cells are then evaluated for CAM expression.

[1010] 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). The plate is aspirated, the medium is removed and 100 μl of 0.1%
Paraformaldehyde-PBS (with Ca ++ and Mg ++) is added to each well. The plate is kept at 4 ° C. for 30 minutes.

The fixative is then removed from the wells and the wells are washed with PBS (+ Ca, Mg)
Wash once with + 0.5% BSA and drain. Do not allow this well to dry. Add 10 μl of diluted primary antibody to test and control wells. Anti-ICAM-1-Biotin, Anti-VACM-1-Biotin
And anti-E-selectin-Biotin at a concentration of 10 μg / ml (0.1 mg
/ Ml stock antibody (1:10 dilution). The cells are incubated for 30 minutes at 37 ° C. in a humidified environment. Wells were washed with PBS (+ Ca, Mg
) Wash three times with + 0.5% BSA.

Next, 20 μl of the diluted ExtraAvidin-Alkaline Ph
Osphotase (1: 5,000 dilution) is added to each well and
And incubate for 30 minutes. Wells were added with PBS (+ Ca, Mg) +0.5
Wash three times with% BSA. One tablet of p-nitrophenol phosphate (p
NPP) is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Triplicate standard wells were prepared with 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 of the standard wells. The plate must be incubated at 37 ° C. for 4 hours. 3M NaO
Add a volume of 50 μl of H 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 is set to indicate the concentration of AP conjugate in each standard well [5.50
ng; 1.74 ng; 0.55 ng; 0.18 ng]. The results are shown as the amount of bound AP conjugate in each sample.

The studies described in this example tested the activity of a polypeptide of the invention.
However, one of skill in the art can readily modify the illustrated studies to test the activity of a polynucleotide (eg, gene therapy), agonist, and / or antagonist of the present invention.

It is clear that the invention can be carried out in other ways than those described in detail in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings, and so are within the scope of the appended claims.

[1089] 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
Is incorporated herein by reference. In addition, both the hard copy of the sequence listing and the corresponding computer readout form submitted herewith are hereby incorporated by reference in their entirety.

Example 53: Assay for Stimulating Bone Marrow CD34 + Cell Proliferation This assay is based on the isolation of human CD34 + based on its ability to grow in the presence of hematopoietic growth factors and expressed in mammalian cells. Polypeptide is CD3
Evaluate the ability to stimulate 4+ cell proliferation.

[1091] Most mature precursors have previously been shown to respond to only one signal. More immature precursors require at least two signals for a response. Thus, to test the effect of a polypeptide on the hematopoietic activity of a wide range of progenitor cells, the assay includes a given polypeptide in the presence or absence of other hematopoietic growth factors. The isolated cells are cultured for 5 days in the presence of stem cell factor (SCF) in combination with the sample to be tested. SCF alone has a very limited effect on bone marrow (BM) cell proliferation and acts only as a "survival" factor under such conditions. However, these cells (eg, IL-3)
SCF in combination with any factor exhibiting a stimulatory effect in causes a synergistic effect. Thus, if the tested polypeptide has a stimulatory effect on hematopoietic precursors, such activity can be easily detected. Normal BM cells have low levels of circulating cells and thus do not appear to detect any inhibitory effects of a given polypeptide, or an agonist or antagonist thereof. Thus, assays for inhibitory effects on precursors are preferably first subjected to in vitro stimulation with SCF + IL + 3 and then tested in cells that have been contacted with the compound to be evaluated for inhibition of such induced proliferation.

[1025] Briefly, CD34 + cells are isolated using methods known in the art. The cells are thawed and medium (QBSF containing 1% L-glutamine)
60 Serum-free medium (500 ml) Quality Biological, I
nc. , Gaithersburg, MD Cat # 160-204-101)
Resuspended in. After several gentle centrifugation steps at 200 xg, the cells are left to stand for 1 hour. The cell number is adjusted to 2.5 × 10 ⁵ cells / ml. At this time, 100 μl of sterile water is added to the peripheral wall of the 96-well plate. The cytokines that can be tested with a given polypeptide in this assay are rng at 50 ng / ml.
hSCF (R & D Systems, Minneapolis, MN, Cat #
255-SC) alone, and rhSCF and rhIL-3 at 30 ng / ml.
(R & D Systems, Minneapolis, MN, Cat # 203-
ML). After 1 hour, 10 μl of the prepared cytokine, 5
0 μl SID (1: 2 dilution of the supernatant = 50 μl) and 20 μl of the diluted cells are added to the medium already present in the wells, bringing the final total volume to 100 μl. The plate is then placed in an incubator at 37 ° C./5% CO ₂ for 5 days.

[1097] Eighteen hours prior to the assay, harvest and add 0.5 μCi / well [3H] thymidine to each well in a volume of 10 μl to determine the growth rate. This experiment was performed by To
Harvest cells from each 96-well plate using mtec Harvester 96 and stop by passing through filter mat. After collection, the filter mat is dried, cut, and placed in an OmniFilter assembly consisting of one OmniFilter plate and one OmniFilter tray. 60 μl of Microscint is added to each well and TopS
eal-A press-on sealing film A bar co
This plate, sealed with de 15 sticker, is affixed to the first plate for counting. The sealed plate was then loaded and packed.
The level of radioactivity was determined via ard Top Count, and the printed data was collected for analysis. The level of radioactivity reflects the amount of cell growth.

The study described in this example tests the activity of a given polypeptide to stimulate bone marrow CD34 + cell proliferation. One of skill in the art can readily modify this exemplary study to test the activity of polynucleotides (eg, gene therapy), antibodies, agonists, and / or antagonists and fragments and variants thereof.
As a non-limiting example, potential antagonists tested in this assay inhibit cell proliferation in the presence of cytokines and / or increase inhibition of cell proliferation in the presence of cytokines and certain polypeptides Expect that. In contrast, we expect that potential agonists tested in this assay will promote cell growth and / or reduce inhibition of cell growth in the presence of cytokines and certain polypeptides.

This ability of the gene to stimulate proliferation of bone marrow CD34 + cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of disorders affecting the immune and hematopoietic systems. . Representative uses are described in the above sections “Immunological activity” and “Infectious diseases”, and elsewhere herein.

[1096]

[Table 6] ATCC Accession Number 203960 (Canada) Applicant will file a Canadian Patent until the Canadian Patent is issued or the application is rejected or abandoned and cannot be reinstated or withdrawn. ) Request that only an independent expert designated by the Commissioner be provided with a sample of the deposited biological material referred to in the application, and the applicant Before completion of the regulatory preparations, the International Bureau must be so informed in writing.

(Norway) The applicant hereby assumes that, until the application has been published (by the Norwegian Patent Office) or has not been published and has been decided by the Norwegian Patent Office, the donation of samples shall be Request that it be done only for the house. A request to this effect shall be filed by the applicant with the Norwegian Patent Office before the time when the application is made publicly available under sections 22 and 33 (3) of the Norwegian Patent Act. If such a request is made by the applicant, any request for the provision of a sample by a third party shall indicate the expert employed. The expert is a list of certified experts prepared by the Norwegian Patent Office (list of
Recognized experts), or
In each case, it can be any person approved by the applicant.

[1098] (Australia) The applicant hereby gives the sample of the microorganism, prior to the grant of the patent,
Alternatively, prior to lapsing, rejection or withdrawal of the application, the subject is a skilled adder who has no interest in the invention.
ssee) (Australia Patent Law 3.25 (3)).

(Finland) The applicant hereby filed that the application was (Patent and Control Committee (National)
Until published (by the Board of Patents and Regulations) or without publication and subject to a decision by the National Patent and Legislative Commission, the sample will be provided only to experts in the art. ,Claim.

[1100] (UK) Applicant now claims that a sample of the microorganism will be made available only to professionals. Such a request must be made by the applicant to the International Bureau before the regulatory preparations for international publication of the application have been completed. ATCC accession number 203960 (Denmark) The applicant hereby assumes that until the application has been published (by the Danish Patent Office) or has received a decision by the Danish Patent Office without publication, Request that this be done only to experts. A request to this effect shall be made by the applicant to the Danish Patent Office prior to the time the application is made publicly available under sections 22 and 33 (3) of the Danish Patent Act. If such a request is made by the applicant, any request for the provision of a sample by a third party shall indicate the expert employed. The expert is a list of certified experts prepared by the Danish Patent Office (list of
Recognized experts), or
In each case, it can be any person approved by the applicant.

(Sweden) The applicant hereby assumes that, until the application has been published (by the Swedish Patent Office) or has not been published and has been decided by the Swedish Patent Office, the donation of samples is Request that it be done only for the house. A 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 Application's Gui).
Format PCT / RO / 13 described in annex Z of Volume I of de
4). If such a request is made by the applicant, any request for the provision of a sample by a third party shall indicate the expert employed. The expert is a list of accredited experts created by the Swedish Patent Office (list
to any of the recognized experts), or
Alternatively, it may be any person approved by the applicant in each case.

(Netherlands) The applicant hereby states that until the date of issue of the Dutch patent, or until the date on which the application is rejected, withdrawn or abandoned (lapped), the microorganisms must be specialized under the provisions of Patent Act 31F (1). Request that this be done only in the form of sample donation to the house. The request to this effect shall be filed by the applicant prior to the date on which the application is made publicly available under section 22C or 25 of the Dutch Patent Law, whichever comes first. Shall be submitted to the Bureau.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 48/00 A61P 7/02 4C085 A61P 3/10 7/04 4C087 7/02 7/06 4H045 7/04 11/00 7/06 11/06 11/00 13/12 11/06 17/00 13/12 25/00 17/00 27/02 25/00 29/00 27/02 101 29/00 31/18 101 37/00 31/18 37/02 37/00 C07K 14/47 37/02 16/18 C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/02 5/10 1/68 A C12P 21/02 G01N 33/53 D C12Q 1/02 M 1/68 33/566 G01N 33/53 C12N 15/00 ZNAA 5/00 A 33 / 566 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, M , NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID , IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, T R, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Rosen, Craig A. United States Maryland 20882, Layton'sville, Rolling Hill Road 22400 (72) Inventor Leuven, Stephen M. United States Maryland 20882, Layton'sville, Heritage Hills Drive 18528 (72) Inventor Commat Souris, George United States Maryland 20901, Silver Spring, Girwood Street 9518 F-term (reference) 4B024 AA01 AA11 BA44 BA80 CA04 CA09 DA02 DA03 DA06 EA02 EA04 GA03 HA14 HA15 4B063 QA01 QA12 QA18 QA19 QQ12 QQ79 QR32. BA22 CA53 NA14 ZA022 ZA162 ZA332 ZA362 ZA452 ZA532 ZA542 ZA552 ZA592 ZA812 ZA892 ZA942 ZA972 ZB072 ZB082 ZB092 ZB112 ZB132 ZB152 ZB262 ZB272 ZB312 ZB332 ZB352 ZB382 ZC552 4C085 AA13 AA14 DD62 4C087 AA01 AA02 AA03 BC83 CA12 NA14 ZA02 ZA16 ZA33 ZA34 ZA36 ZA45 ZA51 ZA53 ZA54 ZA55 ZA59 ZA66 ZA67 ZA75 ZA81 ZA89 ZA94 ZA96 ZA97 ZB07 ZB08 ZB09 ZB11 ZB13 ZB15 Z B26 ZB27 ZB31 ZB33 ZB35 ZB37 ZB38 ZC55 4H045 AA10 AA11 AA20 CA40 DA76 DA86 EA20 EA50 FA74

Claims (23)

[Claims] 1. An isolated nucleic acid molecule comprising: (a) a polynucleotide fragment of SEQ ID NO: X or a polynucleotide fragment of a cDNA sequence contained in ATCC Deposit No. Z capable of hybridizing to SEQ ID NO: X (B) a polynucleotide encoding a polypeptide fragment of SEQ ID NO: Y or a polypeptide fragment encoded by the cDNA sequence contained in ATCC deposit number Z capable of hybridizing to SEQ ID NO: X; (c) SEQ ID NO: Y A polynucleotide encoding the polypeptide domain of SEQ ID NO: Y, or a polypeptide domain encoded by the cDNA sequence contained in ATCC Deposit No. Z, which is capable of hybridizing to SEQ ID NO: X; Hybrida on number X A polynucleotide encoding a polypeptide epitope encoded by the cDNA sequence contained in ATCC Deposit No. Z; or (e) a polynucleotide encoding a polypeptide of SEQ ID NO: Y having biological activity, or A cDNA sequence contained in ATCC Deposit No. Z that can hybridize to No. X; (f) a polynucleotide that is a variant of SEQ ID No. X; (g) a polynucleotide that is an allelic variant of SEQ ID No. X; A polynucleotide encoding a species homolog of SEQ ID NO: Y; (i) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) to (h), Here, the polynucleotide has a nucleotide sequence consisting of only A residues or only T residues. The nucleic acid molecule does not hybridize under stringent conditions, a polynucleotide, comprising a polynucleotide having at least 95% identical to the nucleotide sequence to a sequence selected from the group consisting of isolated nucleic acid molecules. 2. The isolated nucleic acid molecule of claim 1, wherein said polynucleotide fragment comprises a nucleotide sequence encoding a secreted protein. 3. The method according to claim 2, wherein the polynucleotide fragment encodes a nucleotide sequence encoding the polypeptide identified by SEQ ID NO: Z or a cDNA sequence encoded by the cDNA sequence contained in ATCC accession number Z capable of hybridizing to SEQ ID NO: X. 2. The isolated nucleic acid molecule of claim 1, comprising. 4. The isolated polynucleotide of claim 1, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO: X or the cDNA sequence contained in ATCC accession number Z capable of hybridizing to SEQ ID NO: X. Nucleic acid molecule. 5. The isolated nucleic acid molecule of claim 2, wherein said nucleotide sequence comprises a contiguous nucleotide deletion from either the C-terminus or the N-terminus. 6. The isolated nucleic acid molecule of claim 3, wherein said nucleotide sequence comprises a contiguous nucleotide deletion from either the C-terminus or the N-terminus. 7. A recombinant vector comprising the isolated nucleic acid molecule of claim 1. 8. A method for producing a recombinant host cell comprising the isolated nucleic acid molecule of claim 1. 9. A recombinant host cell produced by the method of claim 8. 10. The recombinant host cell of claim 9, comprising a vector sequence. 11. An isolated polypeptide, comprising: (a) a polypeptide fragment of the encoded sequence contained in SEQ ID NO: Y, or ATCC Deposit No. Z; (C) a polypeptide domain of the encoded sequence contained in SEQ ID NO: Y or ATCC accession number Z; (c) a polypeptide domain of the encoded sequence contained in SEQ ID NO: Y or ATCC accession number Z; d) a polypeptide epitope of SEQ ID NO: Y or the encoded sequence contained in ATCC Deposit No. Z; (e) a secreted form of SEQ ID NO: Y or the encoded sequence contained in ATCC Deposit No. Z; A) a full-length protein of SEQ ID NO: Y, or the encoded sequence contained in ATCC Deposit No. Z; (g) a variant of SEQ ID NO: Y; Allelic variants of SEQ ID NO: Y; or (i) SEQ ID NO: Y of species homologs, including at least 95% identical to the amino acid sequence to a sequence selected from the group consisting of,
An isolated polypeptide. 12. The isolated polypeptide of claim 11, wherein said secreted form or said full length protein comprises a continuous amino acid deletion from either the C-terminus or N-terminus. 13. An isolated antibody that specifically binds to the isolated polypeptide of claim 11. 14. Expressing the isolated polypeptide of claim 11,
Recombinant host cells. 15. A method for producing an isolated polypeptide, comprising: (a) culturing the recombinant host cell of claim 14 under conditions such that the polypeptide is expressed. And (b) recovering the polypeptide. 16. A polypeptide produced by the method of claim 15. 17. A method for preventing, treating, or alleviating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of a polypeptide of claim 11 or a polynucleotide of claim 1. Administering to the body. 18. A method for diagnosing a pathological condition or susceptibility to a pathological condition in a subject, comprising: (a) determining the presence or absence of a mutation in the polynucleotide of claim 1 Diagnosing a pathological condition or susceptibility to the pathological condition based on the presence or absence of the mutation. 19. A method for diagnosing a pathological condition, or susceptibility to a pathological condition in a subject, comprising: (a) the presence of expression of the polypeptide of claim 11 in a biological sample. Or determining the amount, and (b) diagnosing a pathological condition or susceptibility to the pathological condition based on the presence or amount of expression of the polypeptide. 20. A method for identifying a binding partner to a polypeptide according to claim 11, comprising: (a) contacting the polypeptide according to claim 11 with a binding partner;
And (b) determining whether the binding partner results in the activity of the polypeptide. 21. A gene corresponding to the cDNA sequence of SEQ ID NO: Y. 22. A method for identifying activity in a biological assay, comprising:
Wherein the method comprises the steps of: (a) expressing SEQ ID NO: X in cells; (b) isolating the supernatant; (c) detecting activity in a biological assay; and (d) C) identifying a protein in said supernatant having said activity. 23. A product produced by the method of claim 20.