JP2003523733A - 10 human secretory proteins - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel human secreted proteins and isolated nucleic acids comprising the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies for producing human secreted proteins, as well as recombinant methods for producing human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating diseases, disorders and / or conditions related to these novel human secreted proteins.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to newly identified polynucleotides, polypeptides encoded by these polynucleotides, antibodies that bind to these polypeptides, such polynucleotides, polypeptides, and It relates to the use of antibodies, as well as their production.

BACKGROUND OF THE INVENTION Unlike bacteria, which exist as a single compartment surrounded by a membrane, human cells and other eukaryotic cells are subdivided by the membrane into many functionally distinct compartments. Each membrane-separated compartment, or organelle, contains different proteins that are essential for the function of that organelle. Cells use "sorting signals," which are amino acid motifs located inside proteins, to target the protein to specific cellular organelles.

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

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

Despite significant advances in recent years, only a few genes encoding human secretory 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 widespread role of secreted proteins in human physiology, there is a need to identify and characterize new human secreted proteins and the genes that encode them. This finding makes it possible to detect, treat and prevent medical diseases, disorders and / or conditions by using secreted proteins or the genes encoding them.

SUMMARY OF THE INVENTION The present invention relates to novel polynucleotides and their encoded polypeptides. Further, the invention relates to vectors, host cells, antibodies and recombinant and synthetic methods for producing the polypeptides and polynucleotides.
Also provided are diagnostic methods for detecting disorders and / or conditions associated with the polypeptides and polynucleotides, as well as therapeutic methods for treating such diseases, disorders and / or conditions. The invention further relates to screening methods for identifying binding partners of this 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 taken from its original environment (eg, the natural environment if it is naturally occurring), and thus It has been altered "by the hand of man" from its natural state. For example, an isolated polynucleotide can be part of a vector or composition of matter, or contained within 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
Libraries, whole cells or mRNA preparations, genomic DNA preparations (
Sheared whole cell genomic DNA preparations, including those separated by electrophoresis and transferred to a blot, or other compositions that the art does not exhibit the distinguishing characteristics of the polynucleotides / sequences of the invention. I don't say anything.

In the present invention, “secreted” proteins are proteins that can be directed as a result of a signal sequence in the ER, secretory vesicles, or extracellular space, as well as those that do not necessarily contain a signal sequence but are released into the extracellular space. Refers to protein. If a secreted protein is released into the extracellular space, it may undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.

In a particular embodiment, 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, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.
It is 5 kb, 5 kb, 2.5 kb, 2.0 kb or 1 kb or less. In a further embodiment, the polynucleotide of the invention, as disclosed herein, comprises part of the coding sequence but not all or part of any intron. In another embodiment, the polynucleotide comprising the coding sequence does not include the coding sequence of a genomic flanking gene (ie, 5'or 3'to the gene of interest in the genome). In another embodiment, the polynucleotide of the invention is 10
00, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3,
Does not contain more than 2 or 1 genomic flanking gene coding sequences.

As used herein, “polynucleotide” refers to SEQ ID NO: X (SE
Q ID NO: X) refers to a molecule having the nucleic acid sequence contained in it or a cDNA contained in a clone deposited with the ATCC. For example, the polynucleotide includes 5'and 3'untranslated sequences, a coding region with or without a signal sequence, a nucleotide sequence of a full-length cDNA sequence containing a secretory protein coding region, and fragments, epitopes, domains of this nucleic acid sequence. , And variants. Further, as used herein, "polypeptide" refers broadly 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 duplicating sequences contained in multiple clones (contig analysis).
A representative clone containing all or most of the sequence for SEQ ID NO: X has been deposited with the American Type Culture Collection ("ATCC"). As shown in Table 1, each clone is identified by its cDNA clone ID (identifier) and ATCC Deposit Number. ATCC is 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 Prosecution Purposes.

The “polynucleotide” of the present invention is also capable of hybridizing under stringent hybridization conditions to the sequence contained in SEQ ID NO: X, its complement, or the cDNA in the clone deposited with the ATCC. Contains nucleotides. “Stringent hybridization conditions” means 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. It refers to washing the filter at about 65 ° C. in.

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

Note that the changes in the above conditions can be achieved by the inclusion and / or substitution of alternative blocking reagents used to suppress background in hybridization experiments. 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 poly A + sequence (eg, any 3 ′ end poly A + region of the cDNA shown in the sequence listing) or a poly hybridizing only to a complementary stretch of T (or U) residues. Nucleotides are not included in the definition of "polynucleotide." Because such a polynucleotide hybridizes 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). Because it soybeans.

The polynucleotide of the present invention may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or unmodified DN.
It may be A or modified RNA or modified DNA. For example, a polynucleotide is a DNA that is a mixture of single-stranded and double-stranded DNA, single-stranded and double-stranded regions.
Single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, single-stranded, or more typically double-stranded or a mixture of single-stranded and double-stranded regions. It can be composed of 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 may 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; thus, "
A "polynucleotide" includes chemically, enzymatically, or metabolically modified forms.

The polypeptides of the present invention may be composed of amino acids linked to each other by peptide bonds or modified peptide bonds, ie, peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. May be included. The polypeptide is either by a natural process, such as post-translational processing, or by chemical modification techniques well known in the art.
It can be modified. Such modifications are based on basic texts and more detailed research papers,
And well described in many research literatures. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides can be branched, eg, as a result of ubiquitination, and polypeptides can be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides can result from natural post-translational processes or can be made by synthetic methods. As a modification,
Acetylation, acylation, ADP-ribosylation, amidation, covalent binding of flavins,
Covalent bond of heme moiety, covalent bond of nucleotide or nucleotide derivative, covalent bond of lipid or lipid derivative, phosphatidylinositol
covalent bond, cross-linking, cyclization, disulfide bond formation,
Demethylation, covalent crosslink formation, cysteine formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Pegylatio
n), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (For example, PROTEI
NS-STRUCTURE AND MOLECULAR PROPERTIE
S. Second Edition, T.S. E. Creighton, W.C. 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 NY A.
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, both sequences being , Identified by the integers specified in Table 1.

A “polypeptide having biological activity” refers to a polypeptide of the invention (mature form (Including), but not necessarily the same. If a dose dependence is present, it need not be identical to that of this polypeptide, but rather is substantially similar to that of a given activity when compared to a polypeptide of the invention ( That is, the candidate polypeptide is
Shows greater activity or about 1/25 compared to the polypeptides of the invention
More, and preferably about 1/10 or more of activity, and most preferably about 1/3
The above activity is shown).

(Polynucleotides and Polypeptides of the Present Invention) (Characteristics of Protein Encoded by Gene No. 1) As a non-limiting example, the translation product of this gene has the accession number gb | AAC22346.1 of the following database. Sequences accessible through (all information available through listed accession numbers, incorporated herein by reference) (
Here, this is "| glycerol-3-phosphotase tra
nsporter (glpT) [Haemophilus influenzae
e Rd] ”).

In another embodiment, a polypeptide comprising the amino acid sequence of the open reading frame upstream of the putative signal peptide is contemplated by the present invention.

In a particular embodiment, the polypeptide of the invention has the following amino acid sequence: S
RGDLFALGSGIIAYGFSKFIMGGSVSDRSNPRVFLP
AGLILAAAVMLFMGFVPWATSSIAVMFVLLFLCGWF
QGMGWPPCGRTMVHWWWSQKERGGIVSVWNCAHNVGG
GIPPLLLFGMAWFNDWHAALYMPAFCAILVALFAF
AMMRDTPQSCGLPPIEEYKNDYPDDYNEKAEQELTA
It contains or consists of an amino acid sequence such as KQPGGRLRLHPHAYTAA (SEQ ID NO: 61). Further, fragments and variants of these polypeptides (eg, the fragments described herein, at least 80%, 85%, 90%, 95%, 96%, 97% of these polypeptides,
Polypeptides encoded by polynucleotides that are 98%, or 99% identical, and polynucleotides that hybridize to the polynucleotides encoding these polypeptides under stringent conditions are encompassed by the invention. Antibodies that bind to the polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

[0024]   This gene is mainly expressed in the Spinal cord (spinal cord).

Accordingly, the polynucleotides and polypeptides of the present invention are used for the differential identification of tissues or cell types present in a biological sample and as follows: spinal cord injury and neurodegenerative disease states, including spinal cord. It is useful as a reagent for the diagnosis of diseases and conditions including but not limited to disorders of Similarly, polypeptides and antibodies to these polypeptides are useful in providing immunological probes for the differential identification of tissues or cell types. Significantly higher than standard gene expression levels (ie, expression levels in normal tissues or fluids from individuals without the disorder), or more for many disorders of the tissues or cells, particularly the nervous system Low levels of expression of this gene may result in specific tissue or cell types (eg, neural tissue, cancerous tissue and wound tissue) or body fluids (eg, lymph, serum, plasma, etc.) taken from individuals with such disorders. Urine, synovial fluid and spinal fluid), or in another tissue or sample.

The tissue distribution of this sequence predicts that this gene will be useful in the diagnosis and treatment of spinal cord injury and neurodegenerative disorders. In particular, polynucleotides and polypeptides of this gene, and antibodies to this polypeptide, are useful in the detection and / or treatment of spinal neurodegenerative diseases such as amyotrophic lateral sclerosis and multiple sclerosis. Is considered to be. Furthermore, the translation product of this gene may have use in spinal cord repair after traumatic injury.

More generally, the polynucleotides and / or polypeptides of the invention are
It may be useful in the detection, treatment and / or prevention of neurological disorders including: Alzheimer's disease, Parkinson's disease, Huntington's disease, Tourette's syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathy, neoplasia, Trauma, birth defects, spinal cord injury,
Ischemia and infarction, aneurysm, bleeding, schizophrenia, mania, dementia, paranoia, obsessive-compulsive disorder, depression, panic disorder, learning disability, psychosis, autism, and behavioral changes (nutrition, sleep patterns, Balance, and impaired perception). In addition, this protein is also used to determine its biological activity, in addition to its use as a nutritional supplement,
It can be used to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, and to identify agents that modulate their interaction. The protein as well as antibodies to this protein may show utility as tumor markers for the tissues listed above and / or targets for immunotherapy.

(Characteristics of Protein Encoded by Gene No. 2) This gene is mainly expressed in CD34 + cell, I, frac. II. Human
Ovarian Cancer Reexcision; Fron
It is expressed in tal cortex, epileptic, re-excision.

Accordingly, the polynucleotides and polypeptides of the present invention are used for the differential identification of tissues or cell types present in a biological sample, and in the following: cancer and other proliferative disorders, especially of the ovary. It is useful as a reagent for the diagnosis of diseases and conditions including, but not limited to. Similarly, polypeptides and antibodies to these polypeptides are useful in providing immunological probes for the differential identification of tissues or cell types. Significantly higher than normal gene expression levels (ie expression levels in normal tissues or fluids from individuals without the disorder), or more for many disorders of the tissues or cells, especially the reproductive system Low levels of expression of this gene may result in specific tissue or cell types (eg, cancerous and wound tissue) or body fluids (eg, lymph, serum, plasma, urine, synovium, etc.) taken from individuals with such disorders. Fluid and spinal fluid), or in another tissue or sample.

The tissue distribution in CD34 + cells and ovarian cancer indicates that the translation product of this clone may be useful in the study and treatment of hematopoietic disorders and neoplasms.
Secreted proteins also identify agents that modulate their interactions to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to isolate cognate ligands or receptors. Can be used as a supplement and as a nutritional supplement. This product may also have a very broad range of biological activities. Typical applications include the chemotaxis and binding activity sections below, Example 11,
12, 13, 14, 15, 16, 18, 19 and 20, as well as elsewhere herein. Briefly, this protein may have the following activities: induction of cytokines, cell growth / differentiation regulatory activity, or other cytokines; immunostimulatory / immunosuppressive activity (eg, human immunodeficiency virus infection, cancer. , To treat autoimmune diseases, and allergies; regulate hematopoiesis (eg, to treat anemia or as an adjunct to chemotherapy); stimulate or proliferate bone, cartilage, tendons, ligaments, and / or nerves. (For example, to treat wounds, to stimulate follicle-stimulating hormone (to control fertility); chemotactic and chemokinetic activities (
For example, to treat infections, tumors); hemostatic or thrombolytic activity (eg, to treat hemophilia, myocardial infarction, etc.); anti-inflammatory activity (eg, septic shock,
To treat Crohn's disease); as an antimicrobial agent; to treat psoriasis or other hyperproliferative disorders; to regulate metabolism and behavior. Also contemplated is the use of the corresponding nucleic acid in gene therapy procedures. The protein and antibodies to this protein may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of Protein Encoded by Gene No. 3) Preferred polypeptides of the present invention include a polypeptide having an amino acid sequence shown as SEQ ID No. 62 in the sequence listing.

The polypeptide of this gene has been determined for this gene to have a transmembrane domain at amino acid positions approximately 115-131 of the amino acid sequences referenced in Table 1. In addition, the cytoplasmic tail containing amino acids 132-141 of this protein has also been determined. Based on these characteristics, the protein product of this gene is believed to share the structural features of type Ia membrane proteins.

This gene is expressed primarily in the following tissue / cDNA library: S
oares placenta Nb2HP, and to a lesser extent in: Soares_testis_NHT; Soares fatali.
ver spleen 1NFLS; Prostate, BPH, Lib 2;
NCI_CGAP_Pr5; Human colon carinoma (H
CC) cell line, remake; Human Fetal Sple
en; Human Thyroid; Mesangial cell, fra
c 2; Stratagene placenta (# 937225); NCI
_CGAP_Lym12; Gessler Wilms tumor; NCI_
CGAP_Pr2; Stratagene ovarian cancer (#
937219); KMH2; HM1; Hemangiopericytoma;
CHME Cell Line, treated 5 hrs; Colon T
umor; Human Neurophil, Activated; Soa
res_senecent_fibroblasts_NbHSF; NCI_
CGAP_Brn23; Soares_multiple_sclerosis
_2NbHMSP; Soares_placena_8to9weeks_2
NbHP8to9W; T cell helper II, and Soares
infant brain 1 NIB.

Accordingly, the polynucleotides and polypeptides of the present invention are for the differential identification of tissues or cell types present in a biological sample and as follows: including but not limited to placenta and proliferative disorders. It is useful as a reagent for diagnosis of diseases and conditions. Similarly, polypeptides and antibodies to these polypeptides are useful in providing immunological probes for the differential identification of tissues or cell types. Significantly higher than standard gene expression levels (ie expression levels in normal tissues or fluids from individuals without the disorder) for many disorders of the tissues or cells, especially placenta, ovary, and fetus Or a lower level of expression of this gene results in a particular tissue or cell type taken from an individual having such a disorder (eg, female reproductive organs, fetal tissue, cancerous tissue and wound tissue).
Or it can be routinely detected in body fluids such as lymph, serum, plasma, urine, synovial fluid and spinal fluid, or another tissue or sample.

The tissue distribution is such that the polynucleotides and polypeptides corresponding to this gene are
It is shown to be useful in the diagnosis and / or treatment of placental disorders (including placental symbiosis). Specific expression in the placenta suggests that this gene product may play a role in the proper establishment and maintenance of placental function. Alternatively, the gene product is
Produced by the placenta and then transported to the embryo, where this product may play an important role in the development and / or survival of the developing embryo or fetus. Expression of this gene in vascular-rich tissues such as placenta also indicates that this gene product can be produced more commonly in endothelial cells or in the circulation. In such cases, this product may play a more generalized role in vascular functions such as angiogenesis. This product can also be produced in blood vessels and has effects on other cells in the circulation, such as hematopoietic cells. This product may serve to promote the growth, survival, activation, and / or differentiation of hematopoietic cells and other cells in the body. Expression in fetal and cancerous tissues indicates that this protein may play a role in regulating cell division and may have utility in the diagnosis and treatment of cancer and other proliferative disorders. Similarly, embryonic development also depends on decisions involved in cell differentiation and / or apoptosis in patterning. Therefore, this protein may also be involved in apoptosis or tissue differentiation and may be useful in the treatment of cancer. Furthermore, this protein, in addition to its use as a nutritional supplement, is used to determine its biological activity, to raise antibodies, as a tissue marker, to isolate its cognate ligand or receptor, It can be used to identify agents that modulate the interaction. The protein and antibodies directed against this protein may show utility as tumor markers and / or immunotherapy targets for the tissues listed above.

(Characteristics of Protein Encoded by Gene No. 4) The polypeptide of this gene was determined to have a transmembrane domain at about amino acid positions 24 to 40 of the amino acid sequence referred to in Table 1 for this gene. Has been done. In addition, the cytoplasmic tail containing amino acids 1-23 of this protein has also been determined. Based on these characteristics, the protein product of this gene is
It is believed to share the structural features of type II membrane proteins.

This gene is expressed primarily in the following tissue / cDNA libraries:
Human 8 Week Whole Embryo, and to a lesser extent, in: Frontal cortex, epileptic, r
e-excision; Soares_parathyroid_tumor_
NbHPA; Soares_fetal_heart_NbHH19W; NTE
RA2 + retinoic acid, 14 days; NCI_CGAP
_Kid6; Soares_pineal_ground_N3HPG; Stra
tagene HeLa cell s3 937216; Soares_to
tal_fetus_Nb2HF8_9w; Soares_pregnant_
uterus_NbHPU; STRATAGENE Human skelet
al muscle cDNA library, cat. # 93632. ;
Human Infant Brain; NCI_CGAP_Pr2; Huma
n Testes Tumor; Human Amygdala; NCI_CG
AP_Lu5; Soares_NFL_T_GBC_S1; Human Cer
ebellum, subtraced; Lung, Cancer (40053
13 A3): Invasive Poorly Differentiate
ed Lung Adenocarcinoma ,; Weizmann Off
actuator Epithelium; Stratagene schizo
brain S11; Human Osteosarcoma; Stratag
ene lung carinoma 937218; Gessler Wi
lms tumor; L428; Human Hypothalmus, Sch
izophrenia; Synovial Fibroblasts (cont
Stratagene hNT neuron (# 937233);
Stratagene colon (# 937204); Normal col
on; Human Placenta; human tonsils; Huma
n Microvascular Endothelial Cells, fr
act. A: Soares overly Tumor NbHOT; Activ
aged T-cell (12h) / Thiouridine-re-exci
sion; Nine Week Old Early Stage Human
Soares melanocyte 2NbHM; T cell help
er II; Human Cerebellum; Soares_fetal_
liver_spleen_1NFLS_S1; Primary Dendri
tic Cells, lib 1; Soares infant brain
1NIB; Soares fatal liver spleen 1NFLS
Human Human Liver; NCI_CGAP_Brn52; Ea
rly Stage Human Brain, random primed;
Larynx carinoma II; Prostate, BPH, Lib
2; Colon Normal; Larynx Tumour; Human
Adult Heart; Johnston frontal cortex;
CD34 + cell, I, frac II; Human White Fat
Human aorta polyA + (TFujiwara); Saos2
Cells, Vitamin D3 Treated; Normaliz
ed Feetal Liver, II; Palate normal; NCI_
CGAP_GC5; Human Adult Pulmonary; NCI_C
GAP_Ov23; Epididymus, caput & corpu
S. Amniotic Cells-TNF induced; cer
ebellum, Enzyme subtracted; Human Lung
NCI_CGAP_Ut4; Human retina cDNA rand
omly primed subbryary; HEL cell line
Breast Cancer cell line, MDA 36; H Fe
male Blader, Adult; Synovial hypoxia;
Jurkat T-Cell, S phase; NCI_CGAP_Lym12
; Wilm's Tumor; Human Manic Depression
Tissue; NCI_CGAP_Alv1; Human Neuroph
il; KMH2; NCI_CGAP_Ut1; Apoptotic T-cel
l; Human Umbilical Vein Endothelial C
ells, unduced; Human Fetal Dura Mate
r; Human Heart; Human umbilical vein
ndothelial cells, IL-4 induced; Human
Hippocampus; Olfactories epithelium, nas
alcavity; Human Activated T-Cells, re-
excision; NCI_CGAP_CLL1; Human Fetal B
rain; Human Thymus Stromal Cells; CHME
Cell Line, treated 5 hrs; Human Whole
Six Week Old Embryo; Macrophage-oxLD
L, re-excision; NCI_CGAP_Co3; Human Gal
I Bladder; Neurophils IL-1 and LPS i
nduced; Human Eosinophils; Human Place
nta; Adipocytes; Human Fetal Lung III;
Human Synovial Sarcoma; Human Ovarian
Cancer Reexcision; Human Testes, Reex
condition; Human Neutrophil, Activated; NC
I_CGAP_Kid5; NCI_CGAP_Brn23; Human Bon
e Marrow, treated; Bone Marrow Cell Li
ne (RS4, 11); Human Endometric Tumor; H
odgkin's Lymphoma II; Keratinocyte; So
ares_fetal_long_NbHL19W and Soares_tes
tis_NHT.

Accordingly, the polynucleotides and polypeptides of the present invention are for the differential identification of tissues or cell types present in a biological sample and as follows: diseases including but not limited to disorders of development and proliferation. And are useful as reagents for diagnosing conditions. Similarly, polypeptides and antibodies to these polypeptides are useful in providing immunological probes for the differential identification of tissues or cell types. For many disorders of the above tissues or cells, particularly developing and / or proliferating tissues, to standard gene expression levels (ie, expression levels in healthy tissues or body fluids from individuals without the disorder). Significantly higher or lower levels of expression of this gene are associated with specific tissue or cell types (eg, fetal tissue, neural tissue, cancerous tissue and wound tissue) taken from an individual with such a disorder. ) Or body fluids (eg lymph, serum, plasma, urine, synovial fluid and spinal fluid)
, Or in another tissue or sample, can be routinely detected.

Furthermore, the supernatant from transient transfection with an expression plasmid containing this gene has been demonstrated to have biological activity on immune / hematopoietic cells. Specifically, the induction of GAS elements is described in Jurkat E and U937.
It was found on both cells (both T cells) and calcium flux was initiated on neutrophils. While calcium flux is an indicator of signal transduction activity, activation of GAS elements indicates the presence of interferon gamma-like activity. Therefore, it can be reasonably concluded that the protein product of this gene is involved in immune regulation.

Expression in fetal heart and brain indicates that the product of this clone may be useful in the study and treatment of impaired cardiovascular and nervous system development and function, as well as some neurodegenerative diseases. . Furthermore, the expression of this gene in fetal and cancer cells, and its overt activity in immune cells, suggests that the protein product of this gene acts as a developmental signal and / or immunomodulator. Therefore, this product may be useful in the diagnosis and treatment of cancer and other proliferative disorders. In addition, the protein product of this gene is associated with immunodeficiency diseases (eg AI
DS) It may be useful in the diagnosis and treatment of age-related immunosuppression and in the treatment of immunodeficiency caused by chemotherapy or radiation therapy. Also, the protein product of this gene may be useful in the treatment of autoimmune disorders such as lupus.
In addition, the protein, in addition to its use as a nutritional supplement, also for isolating cognate ligands or receptors, as tissue markers, to elicit antibodies, to determine biological activity. , Can be used to identify agents that modulate their interaction. The protein as well as antibodies to this protein may show utility as tumor markers for the tissues listed above and / or targets for immunotherapy.

(Characteristics of Protein Encoded by Gene No. 5) Using the computer algorithm BLASTX, the translation product of this gene is, as a non-limiting example, accession number emb | 42901.
1 (all information available through the listed accession numbers is hereby incorporated by reference) through a sequence (which is referred to herein as "(AL03
3377) dJ287G14.2 (PUTATIVE novel seven)
transmembrane protein) [Homo sapiens
]))). Based on structural similarities, these homologous polypeptides are expected to share at least some biological activity; in particular, this sequence is a signal transduction cross-membrane (
signal transduction cross membrane)
Seems to be involved in. Such activities are known in the art, and some of them are described elsewhere herein. Assays for determining such activity are also known in the art, some of which are described elsewhere herein.

Preferred polypeptides of the invention include the polypeptide having the amino acid sequence set forth in SEQ ID NO: 64 in the Sequence Listing.

The polypeptide of this gene has been determined to have a transmembrane domain at approximately amino acid positions 29-45 and 143-159 of the amino acid sequences cited in Table 1 for this gene. Based on these features, the protein product of this gene is believed to share structural features with type IIIa membrane proteins.

This gene is expressed primarily in immune cells and in tissues that tend to attract immune cells (ie, tumors). Specifically, this transcript is found in the following tissue / cDNA library: Human Eosinoph.
ils; NCI_CGAP_GCB1, and to a lesser extent in:
NCI_CGAP_Co8; NCI_CGAP_Sub3; NCI_CGAP_
Co14; B-cells (unstimulated); NCI_CGAP
_Lym12; Human Fetal Brain; Colon Tumor
NCI_CGAP_GC4; NCI_CGAP_GC6; T Cell he
Lper I; Hodkin's Lymphoma II; Activate
d T-cell (12h) / Thiouridine-re-excisio
n; Soares_fetal_heart_NbHH19W; Spleen,
Chronic lympholytic leukemia, and Soar
es_testis_NHT.

Accordingly, the polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample, and in the following: cancer and other proliferative disorders, particularly colon cancer and leukemia. It is useful as a reagent for the diagnosis of diseases and conditions including, but not limited to. Similarly, polypeptides and antibodies to these polypeptides are useful in providing immunological probes for the differential identification of tissues or cell types. Significantly higher than standard gene expression levels (ie expression levels in normal tissues or fluids from individuals without the disorder) for many disorders of the tissues or cells, particularly the colon and immune system, Or a lower level of expression of this gene results in a specific tissue or cell type (eg, cancerous and wound tissue) or body fluid (eg, lymph, serum, plasma, urine) taken from an individual having such a disorder. , Synovial fluid and spinal fluid), or another tissue or sample.

Expression in eosinophils and other immune cell types and homology to secreted G protein-coupled receptors are useful in the study and treatment of inflammation, systemic immune and hematopoietic disorders, and proliferative disorders, particularly colon cancer and leukemia. Suggest sex. In addition, the protein, in addition to its use as a nutritional supplement, also for isolating cognate ligands or receptors, as tissue markers, to elicit antibodies, to determine biological activity. , Can be used to identify agents that modulate their interaction. The protein and antibodies to this protein are
It may show utility as a tumor marker for the tissues listed above and / or as a target for immunotherapy.

Characterization of Protein Encoded by Gene No: 6 This gene is expressed primarily in the following tissue / cDNA libraries:
Human Neurophil; Human Whole Six Wee
k Old Embryo.

When tested against the CTLL / SRE lymphocyte cell line, the protein product of this gene gave a positive result in the SEAP reporter assay. A positive SEAP assay is an indicator of growth factor or cytokine induced cell stimulation.
This SEAP assay incorporates the use of SRE (serum response element) and is typically involved in upregulation of cell function, growth and / or proliferation, eg, in many different cell types when bound by serum response factors. Leads to activation of gene expression. Thus, the protein product of this gene is expected to be useful in upregulating activation, growth and / or proliferation of immune cells, particularly but not limited to neutrophils and T cells.

The polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample, and include, but are not limited to, immunological, developmental and proliferative disorders. It is useful as a reagent for diagnosis of non-limiting diseases and conditions. Similarly, polypeptides and antibodies to these polypeptides are useful in providing immunological probes for the differential identification of tissues or cell types. Significantly relative to standard gene expression levels (ie, expression levels in normal tissues or body fluids from individuals without the disorder) for many disorders of the tissues or cells, particularly the immune system and developing embryo. Higher or lower levels of expression of this gene result in specific tissues or cell types taken from individuals with such disorders (eg, white blood cells, red blood cells, cancerous tissues and wound tissues).
Or it can be routinely detected in bodily fluids such as serum, plasma, urine, synovial fluid and spinal fluid, or another tissue or sample.

Expression in embryonic tissue indicates that this protein may play a role in the regulation of cell division, and the diagnosis, treatment, and / or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. It shows that it can be useful in. Exemplary applications are described in the "Hyperproliferative Disorders" and "Regeneration" sections below, and elsewhere herein. Briefly, developing tissue relies on decisions regarding cell differentiation and / or apoptosis in patterning. Dysregulation of apoptosis can result in inadequate suppression of cell death as occurs in the development of some cancers, or acquired immunodeficiency and certain neurodegenerative disorders such as spinous muscular atrophy (SMA). A), the degree of cell death may not be controlled. Alternatively, the gene product may be involved in a pattern of cell proliferation with early embryonic development. Thus, aberrant expression of this gene product in tissues, particularly adult tissues, may be associated with aberrant cell growth patterns such as found in various cancers. Due to its potential role in proliferation and differentiation, this gene product may have application in adults for tissue regeneration and treatment of cancer. It can also act as a morphogen to control cell and tissue type specification. Accordingly, the polynucleotides and polypeptides of the present invention include the above disorders and conditions,
As well as in the treatment, detection and / or prevention of other types of degenerative conditions. Thus, the protein may regulate apoptosis or tissue differentiation and is useful in detecting, treating, and / or preventing degenerative or proliferative conditions and diseases. Because of this tissue distribution in neutrophils, polynucleotides and polypeptides corresponding to this gene are useful in the diagnosis and / or treatment of various immune system disorders including, but not limited to: Adult respiratory distress Syndrome, alcohol hepatitis, radiation injury, emphysema, arthritis, asthma, immunodeficiency disease (eg, AIDS), leukemia, rheumatoid arthritis, granulomatosis, inflammatory bowel disease, sepsis,
Acne, neutropenia, neutropenia, psoriasis, hypersensitivity (eg T cell mediated cytotoxicity); immune response to transplant organs and tissues (eg anti-graft host disease and anti-host) Graft 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. This protein can also be used to gain new insights in the regulation of cell growth and proliferation. In addition, the protein, in addition to its use as a nutritional supplement, to determine biological activity, to raise antibodies, to isolate cognate ligands or receptors as tissue markers, , Can be used to identify agents that modulate their interaction. The protein as well as antibodies to this protein may show utility as tumor markers for the tissues listed above and / or targets for immunotherapy.

(Characteristics of Protein Encoded by Gene No. 7) The gene encoding the disclosed cDNA is considered to be present on chromosome 22. Therefore, the polynucleotide of the present invention is useful as a marker in linkage analysis for chromosome 22.

The polypeptide of this gene has been determined for this gene to have a transmembrane domain at approximately amino acid positions 6-22 and 49-65 of the amino acid sequence referenced in Table 1. Based on these features, the protein product of this gene is believed to share structural features with type IIIb membrane proteins.

In a particular embodiment, the polypeptide of the invention has the following amino acid sequence: M
EKPRSIETPSEPMEEEEDDDLELFGGYDSFRSYN
SSVGSESSSYLEESSEAENE DREAGELPTSPLHLLSPPGTPRSLDGSGSEPAVCEMC
GIVGTREAFFSKTKRFCSVSCSRSSYSSNSKKASILA
RLQGKPPTKKKAKVLHKVPGLPKEPSSNLKGQDSWQ
Contains XGHQQDKTLWSXASTG (SEQ ID NO: 66) or is composed of such an amino acid sequence. In addition, fragments and variants of these polypeptides (eg, fragments described herein, at least 80%, 85%, 90%, 95%, 96%, 97%, 9 relative to these polypeptides).
Polypeptides that are 8%, or 99% identical, and those encoded by polynucleotides that hybridize to the polynucleotides encoding these polypeptides under stringent conditions) are encompassed by the invention. . Antibodies that bind to the polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In another embodiment, a polypeptide comprising the amino acid sequence of the open reading frame upstream of the putative signal peptide is encompassed by the invention.

In a particular embodiment, the polypeptide of the invention has the following amino acid sequence: F
LHLVGETETVSWRSPGVLRRRPHLQNQWRRKRKMTTW
SCLVAMIVSGVITAVWAVRAAPIWRSQVKQKMRIGK
QGNCRPPRCICSALGLLAPWMAVVLSQLSVRCVVSW
VQGKPSSPRPRGSAASPAPGATPPTPRKPVSWLGYR
ENHRPKKPKSXTRCLVXQNWSLPPISKDRTAGXXDT
NRTRRSGLXLRLG (SEQ ID NO: 65) is included or is composed of such an amino acid sequence. Further, fragments and variants of these polypeptides (eg, the fragments described herein, at least 80%, 85%, 90%, 95%, 96%, 97%, 98 relative to these polypeptides). %,
Or polypeptides that are 99% identical, and polypeptides encoded by polynucleotides that hybridize to the polynucleotides encoding these polypeptides under stringent conditions) are encompassed by the invention. Antibodies that bind to the polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

This gene is expressed primarily in the following tissue / cDNA libraries:
NCI_CGAP_GCB1; Soares fatal river spl
een 1NFLS, and to a lesser extent in: Stratage
ne pancreas (# 937208); Human Testes, Re
excision; Human 8 Week Whole Embryo; K
eratinocyte; Soares_NFL_T_GBC_S1; Soar
es_fetal_heart_NbHH19W; Primary Dendr
itic Cells, lib 1; Human Fetal Epithel
ium (Skin); Stratagene endothelial cel
l 937223; Ovarian Tumor 10-3-95; NCI_C.
GAP_Co3; Colon Normal II; human tonsil
s; T Cell helper I; T cell helper II; S
ares infant brain 1NIB; HE6W; Human O
B HOS treated (1 nM E2) fraction I; NC
I_CGAP_GC5; NCI_CGAP_GC2; eosinophil-I
L5 induced; NCI_CGAP_Br1.1; Aorta endo
therial cells + TNF-a; Activated T-cell
s; Messenger cell, frac 2; HEL cell li
ne; Stratagene schizo brain S11; Human
endometrial stromal cells-treated w
ith progesterone; Human Frontal Corte
x, Schizophrenia; Human Osteosarcoma; H
uman Colon, re-excision; Jurkat T-Cell
, S phase; wilm's tumor; Lymph node b
east Cancer; Human Neutrophil; Breast
Cancer Cell line, angiogenic; Gessler
Wilms tumor; NCI_CGAP_Kid6; Human Fet
al Kidney; Human Umbilical Vein Endot
helial Cells, unduced; Human Chondro
sarcoma; Hepatocellular Tumor, re-exci
sion; Human Gall Blader; Fetal Heart;
CHME Cell Line, unprocessed; Human T-Cel
l Lymphoma; Primary Dendritic cells, f
rac 2; Human Fetal Heart; Activated T-
Cell (12hs) / Thiouridine labelled Eco; E
nudothelial cells-control; HUMAN B CEL
L LYMPHOMA; Human Bone Marrow, treated
Human Testes; Nine Week Old Early St
age Human; Soares_fetal_liver_spleen_
1NFLS_S1; Soares_NhHMPu_S1, and Soares_
testis_NHT.

Accordingly, the polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample and as follows: diseases including but not limited to disorders of the immune system. And are useful as reagents for diagnosing conditions. Similarly, polypeptides and antibodies to these polypeptides include
It is useful in providing immunological probes for the differential identification of tissues or cell types.
Significantly higher than standard gene expression levels (ie, expression levels in normal tissues or fluids from individuals without the disorder), or more, for many disorders of the tissues or cells, particularly the immune system Low levels of expression of this gene
Specific tissues or cell types (eg, bone marrow, lymphoid organs, immune cells, cancerous tissues and wound tissues) or body fluids (eg, lymph, serum, plasma, urine, synovial fluid) collected from individuals with such disorders And cerebrospinal fluid), or in another tissue or sample.

The tissue distribution is such that the polynucleotides and polypeptides corresponding to this gene are
It will be useful in the diagnosis and treatment of various immune system disorders. Typical uses are
In the sections "Immune Reactivity" and "Infectious Diseases" below, Examples 11, 13, 14
, 16, 18, 19, 20 and 27, and elsewhere herein. Briefly, this expression results in the expansion of hematopoietic cell lines containing blood stem cells;
Shows a role in the regulation of survival; differentiation; and / or activation. Involvement in the regulation of cytokine production, antigen presentation, or other processes is associated with the treatment of cancer (eg,
(By boosting the immune response). Expression in cells of lymphoid origin indicates that this natural gene product is involved in immune function. Therefore, this product also has arthritis, asthma, immunodeficiency diseases (eg AIDS), leukemia, rheumatoid arthritis, chronic granulomatosis, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophils. Hypersensitivity, psoriasis, hypersensitivity (eg, T cell-mediated cytotoxicity), immune response to transplanted organs and tissues (eg, graft versus host disease and graft versus host disease).
, Or autoimmune disorders (eg autoimmune infertility), lens tissue injury, demyelination,
It is useful as a drug for immunological disorders including systemic lupus erythematosus, drug-induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. In addition, the protein may represent a secretory factor that influences the differentiation or behavior of other blood cells or recruits hematopoietic cells to the site of injury. Thus, this gene product is implicated in the expansion of stem and committed progenitor cells of various blood lineages.
And are believed to be useful in differentiating and / or proliferating various cell types. In addition, this protein also, in addition to its use as a nutritional supplement,
To elicit antibodies to determine biological activity, as a tissue marker,
It can be used to isolate cognate ligands or receptors and to identify agents that modulate their interaction. The protein, as well as antibodies to this protein, may show utility as tumor markers and / or immunotherapeutic targets for the tissues listed above.

(Characteristics of Protein Encoded by Gene No. 8) Using the computer algorithm BLASTX, the translation product of this gene is, by way of non-limiting example, accession number gb | AAC4012 in the database below.
4.1 | A sequence accessible through (all information available through listed accession numbers is incorporated herein by reference), which is referred to herein as "(A
F053130) unconventional myosin MYO15 [
, Described as “Mus musculus”).

In another embodiment, a polypeptide comprising the amino acid sequence of the open reading frame upstream of the putative signal peptide is encompassed by the invention.

In a particular embodiment, the polypeptide of the invention has the following amino acid sequence: N
GNSTXVVGKGGRLQVPVRNSGSTHASGTDPWSPGRG
RSWAHTWCGRGSAGQGCGMSLASWWPSYGRTQMNS
RASVAGPSWLFCSAPFPHCLSYRSHCSSSCLTRPPG
AWQRASTSCWGPWSSRSWPRGPLGPTPRPSWSGWP
DGGGAAWRWMCSPSARSATRPRWSLPGPGSSWLGGS
CRAEAWXRLPGAGLCHCTPXTHGRTWLA ATLCWT
(SEQ ID NO: 69) or comprises such an amino acid sequence.
Furthermore, fragments and variants of these polypeptides (eg, the fragments described herein, at least 80%, 8% relative to these polypeptides).
By polypeptides that are 5%, 90%, 95%, 96%, 97%, 98%, or 99% identical, and polynucleotides that hybridize to the polynucleotides encoding these polypeptides under stringent conditions Encoded polypeptides, etc.) are encompassed by the present invention. Antibodies that bind to the polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In a particular embodiment, the polypeptide of the invention has the following amino acid sequence: M
GTYLVRQGQCRPGLRRNELFSQLVAQLWQNPDEQQSQ
RGWALMAVLLSAFPPLPVLQKPLLKFVSDQAPRGMA
ALCQHKLLGALEQSQLASGATRAHPPTQLEWLAGWR
RGRMALDVFTFSEECYSAEVESWTTGEQLAGWILQS
RGLXAPXRGWSVSLHSXDAWQDLAGCDVFVLDLIXQT
EDWGDPAXTRXYPITPLGSAXAXITLNPGIQEPSLAQ
Contains EPSKFQPQRCPTRDXTXG (SEQ ID NO: 70) or is composed of such an amino acid sequence. In addition, fragments and variants of these polypeptides (eg, the fragments described herein, at least 80%, 85%, 90%, 95%, 96%, 97% of these polypeptides).
, 98%, or 99% identical, and polypeptides encoded by polynucleotides that hybridize to the polynucleotides encoding these polypeptides under stringent conditions) are encompassed by the invention. It Antibodies that bind to the polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

This gene is expressed primarily in the following tissue / cDNA libraries:
Human Eosinophils, and to a lesser extent in:
B-Cells; Human Osteoblasts II and Resti
ng T-Cell Library, II.

Accordingly, the polynucleotides and polypeptides of the present invention are for the differential identification of tissues or cell types present in a biological sample, as well as the following: including musculoskeletal disorders and immunological disorders. It is useful as a reagent for diagnosis of diseases and conditions not limited to. Similarly, polypeptides and antibodies to these polypeptides are useful in providing immunological probes for the differential identification of tissues or cell types. Significantly higher than standard gene expression levels (ie, expression levels in normal tissues or fluids from individuals without the disorder), or more, for many disorders of the tissues or cells, particularly the immune system Low levels of expression of this gene may result in specific tissue or cell types (eg, white blood cells, cancerous tissue and wound tissue) or body fluids (eg, lymph, serum, plasma, urine) taken from individuals with such disorders. , Synovial fluid and spinal fluid), or another tissue or sample.

Expression in osteoblasts, and homology to atypical myosins, shows utility in the study and treatment of autoreceptive or locomotor motility and other musculoskeletal disorders. Tissue distribution in eosinophils and B cells shows that the protein product of this clone is associated with It will be useful in the diagnosis and treatment of such various immune system disorders. In addition, this protein also, in addition to its use as a nutritional supplement, to elicit antibodies to determine biological activity, to isolate cognate ligands or receptors, as tissue markers. To
It can be used to identify agents that modulate their interaction. The protein, as well as antibodies to this protein, may show utility as tumor markers and / or immunotherapeutic targets for the tissues listed above.

(Characteristics of Protein Encoded by Gene No. 9) Using the computer algorithm BLASTX, the translation product of this gene is, as a non-limiting example, accession number gb | AAD3172 in the database below.
7.1 | AF069307_1 | (Any information available through the listed accession numbers is incorporated herein by reference)
This is "| (AF069307) sodium-dependent" here.
multivitamin transporter [Homo sapie
ns] ”).

In a particular embodiment, the polypeptide of the invention has the following amino acid sequence: M
CTSMAVAASVMGGVVQASLSIHGMCGPMLGLFSLG
IVFPFVNWKGALGGLLTGITLSFWVAIGAFIYPAPA
SKTWPLPLSTDQCIKSNVTATAGPPPLSSRPGIADTW
YSISYLYSYAVGCLGCIVAGVIISLITGRQRGEDIQ
PLLIRPVCNLFCFWSKKYKTLCWCGVQHDSGTEQEN
LENGSARKQGAESVLQNGLRRESLVVVPGPYDPKDKS
Contains YNNMAFETTHF (SEQ ID NO: 71) or consists of such an amino acid sequence. Further, fragments and variants of these polypeptides (eg, the fragments described herein, at least 80%, 85%, 90%, 95%, 96%, 97%, 98 relative to these polypeptides). Polypeptides that are%, or 99% identical, and polypeptides encoded by polynucleotides that hybridize to the polynucleotides encoding these polypeptides under stringent conditions) are encompassed by the invention.
Antibodies that bind to the polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

This gene is mainly expressed in human Kidney (human kidney).

Accordingly, the polynucleotides and polypeptides of the present invention are for differential identification of tissues or cell types present in a biological sample, and for: of diseases and conditions including, but not limited to, renal disorders. It is useful as a reagent for diagnosis. Similarly, polypeptides and antibodies to these polypeptides are useful in providing immunological probes for the differential identification of tissues or cell types. Significantly higher or lower than standard gene expression levels (ie, expression levels in normal tissues or fluids from individuals without the disorder) for many disorders of the tissues or cells, particularly the kidney Levels of expression of this gene are specific tissues or cell types (eg, cancerous and wounded tissues) or body fluids (eg, lymph, serum, plasma, urine, synovial fluid) taken from individuals with such disorders. And cerebrospinal fluid), or in another tissue or sample. When tested on a sensory neuron cell line, supernatants removed from cells containing this gene activated the EGR1 assay. Thus, this gene appears to activate sensory neuron cells via signaling pathways. Early growth response 1 (
Early grow response 1) (EGR1) is a promoter associated with specific genes that induce reactivation, various tissues and cell types, leading cells to differentiation and proliferation.

Tissue distribution in the kidney and sodium-dependent multivitamin transporter (sodium-dependent multivitamin tran)
Homology to the liver) indicates utility in the study and treatment of renal and thyroid ionic balance and metabolism, dysfunction and disorders. In detail,
Polynucleotides, polypeptides and antibodies corresponding to this gene are associated with renal failure,
Nephritis, renal tubular acidosis, urinary protein, pyuria, edema, pyelonephritis, hydronephrosis, nephrotic syndrome, crush syndrome, glomerulonephritis, hematuria, renal colic, and renal stones, as well as Wilms tumor disease and congenital disease. It may be useful in the treatment and / or detection of renal diseases, including renal abnormalities such as horseshoe kidney, polycystic kidney disease, and Fanconi syndrome. The ability of this protein to activate the EGR1 assay in sensory neuronal cell lines indicates that polynucleotides and polypeptides related to this gene, and antibodies to polypeptides related to this gene, are useful in controlling cell differentiation and proliferation. Show that you get. In addition, this protein is also used to determine its biological activity, in addition to its use as a nutritional supplement,
It can be used to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, and to identify agents that modulate their interaction. The protein as well as antibodies to this protein may show utility as tumor markers for the tissues listed above and / or targets for immunotherapy.

CHARACTERISTICS OF PROTEIN ENCODED BY GENE NO: 10 In certain embodiments, a polypeptide of the invention has the following: MPWLADLV
QSSEGSLDVLPVQCLCEFLLLHDAVDDAASXEEDEDG
ETRSRRPRSGRGNRSSGTVXPPAGPATGPEADEQTT
CEVLDYFLRRLGSSQVASRVLAMKGLSLVLSEGSLR
XGEEKEPPMEDVGDTDVLQGYQWLLRDLPRLPLFD
SVRSTTALALQQAIHMETDPQTISAYLIYLSQHTPV
EEQAQHSDLALDVARLVVERSTIMSXLXSKLSPSAA
SDAVLSALLSIFXRYVKAHAGRAREGRRSXLXLVRKS
SRTXVFPYAGSKRGKTAQPWQH (SEQ ID NO: 72), and MR
LHVAXEDGQQSAQHGVRRGTRGELRXEXGHDGGALH
DQPGHVQGQVAVLGLLLHRRRVLGQVDQVGADGLGIS
LHVDCLLQGQGCGAPDAVEQRQARQVPQQPLIALQH
ICVPHILLHGGLLLLPXPQAALRKHQRQTLHGQHAG
GHLGGAEAPQEVVQHXTRGLLISFGPSSRSCRRXHG
AAASVASAASAWPSSAPCLALVVLLPGSSSIVHSIVQQE
LAQTLHGQHVQGALAGLYQVRQP GHGLRGGLPLDD
Contains or consists of an amino acid sequence selected from the group consisting of VEEKVRVSAAHKRCXRRRDWLRLFT (SEQ ID NO: 73). Further, fragments and variants of these polypeptides (eg, the fragments described herein, at least 80%, 85%, 90%, 95%, 96%, 97%, 98 relative to these polypeptides). %, Or 99%
Polypeptides that are identical, and those encoded by polynucleotides that hybridize to the polynucleotides encoding these polypeptides under stringent conditions) are encompassed by the invention. Antibodies that bind to the polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

This gene is expressed primarily in the ovary and to a lesser extent in bone marrow, spleen, breast and T cells.

Accordingly, the polynucleotides and polypeptides of the present invention are for the differential identification of tissues or cell types present in a biological sample and as follows: including but not limited to disorders of hematopoiesis, reproductive and immune systems. It is useful as a reagent for diagnosis of non-limiting diseases and conditions. Similarly, polypeptides and antibodies to these polypeptides are useful in providing immunological probes for the differential identification of tissues or cell types. Significant to standard gene expression levels (ie, expression levels in normal tissues or body fluids from individuals without the disorder) for many disorders of the tissues or cells, particularly the hematopoietic, reproductive and immune systems. Higher or lower levels of expression of this gene may be associated with specific tissue or cell types (eg, cancerous and wounded tissue) or body fluids (such as cancerous and wounded tissue) or body fluids collected from individuals with such disorders.
For example, lymph, serum, plasma, urine, synovial fluid and spinal fluid), or another tissue or sample.

This tissue distribution shows that the polynucleotides and polypeptides corresponding to this gene are used to diagnose and / or diagnose reproductive system, hematopoietic and lymphoid organs, immune system disorders, and cancer.
Alternatively, it is indicated to be useful for treatment.

[0075]

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

The cDNA clone ID was deposited on that date and is labeled "ATCC Deposit No. Z.
And the corresponding accession numbers listed under "Dates". 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 (Total NT Seq)” means “gene number (G
ene No) ”refers to the total number of nucleotides in the contig. The deposited clone may contain all or most of these sequences, designated as "5 'nucleotides of clone sequence" and "3' nucleotides of clone sequence" of SEQ ID NO: X (SEQ ID NO: X). It is reflected by the position of the nucleotide that is assigned. SEQ ID NO: X (SEQ ID of putative initiation codon (methionine)
The nucleotide position of ID NO: X) is identified as the "5 'nucleotide of the start codon". Similarly, SEQ ID NO: X (SEQ ID
The nucleotide position of (NO: X) is “5 of the first amino acid of the signal peptide.
Identified as a'nucleotide '.

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

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

SEQ ID NO: X (SEQ ID NO: X) (where X can be any polynucleotide sequence disclosed in the Sequence Listing) and translated SEQ ID NO: Y (SEQ
ID NO: Y), where Y can be any of the polypeptide sequences disclosed in the sequence listing, are sufficiently accurate and are otherwise known for their various uses and the following. Are suitable for the various applications described further in. For example, the sequence number X (SEQ ID NO: X) is the sequence number X (SEQ ID NO: X).
: X) or the cDNA contained in the deposited clone
It is useful for designing nucleic acid hybridization probes that detect
These probes also hybridize to nucleic acid molecules in biological samples,
It enables the various forensic and diagnostic methods of the invention. Similarly, the polypeptide identified by SEQ ID NO: Y (SEQ ID NO: Y) specifically binds to the proteins (including polypeptides and secreted proteins) encoded by the cDNA clones identified in Table 1, for example. It can be used to make antibodies that bind.

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

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

The present invention also provides SEQ ID NO: X (SEQ ID NO: X), SEQ ID NO: Y (SEQ ID NO: X).
ID NO: Y), or the gene corresponding to the deposited clone. The corresponding gene can be isolated according to known methods using the sequence information disclosed herein. Such methods include the steps of 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
and / or species homologues. SEQ ID NO: X (SEQ ID NO: X), SEQ ID NO: Y (S), using procedures known in the art and using the sequences disclosed herein or the information from the clones deposited with the ATCC.
EQ ID NO: Y) or the full length gene, allelic variant, splice variant, full length coding portion, ortholog, and / or species homolog of the gene corresponding to the deposited clone may be obtained. For example, allelic variants and /
Alternatively, species homologs are isolated and identified by making appropriate probes or primers from the sequences provided herein and screening appropriate nucleic acid sources for allelic variants and / or desired homologs. Can be done.

Table 2 shows preferred epitopes included in certain embodiments of the invention, and polypeptide sequences that may be abandoned according to certain embodiments of the invention. The first column is
Refers to each "gene number" listed in Table 1 above. The second column shows the sequence identifier "nucleotide sequence number X" for the polynucleotide sequences disclosed in Table 1. Three
The row shows the sequence identifier "AA SEQ ID NO: Y" for the polynucleotide sequences disclosed in Table 1. Column 4 is SEQ ID NO: 1 and the final nucleotide minus 15
Denotes a unique integer "ntA" that is any integer between and. And the fifth column shows the unique integer "ntB", which is any integer between 15 and the last nucleotide of SEQ ID NO: X. Here, both ntA and ntB correspond to the positions of the nucleotide residues shown in SEQ ID NO: X, and ntB is +14 or more. For each polynucleotide set forth as SEQ ID NO: X, a uniquely defined integer may be substituted for general formula ab, and may be used to describe a polynucleotide that may preferably be excluded from the invention. Column 6 lists the residues that comprise the preferred epitope contained within the polypeptide encoded by each preferred ORF (SEQ ID NO: Y). Identification of potential immunogenic regions is described by Jam.
eson and Wolf ((1988) CABIOS, 4; 181-186); specifically, Genetics C of this algorithm.
computer group (GCG) implementation (program PEPTIDESTR
UCTURE (Wisconsin Package v10.0, Genet)
ics Computer Group (GCG), Madison, Wisc
) Integrated)). This method returns a value for the probability that a given residue will be found on the surface of proteins. Regions with an antigenicity coefficient score of at least 6 amino acids are shown in Table 2 as "preferred epitopes". The polypeptides of the invention may have 1, 2, 3, 4, 5 or 5 or more of the antigenic epitopes containing the residues listed in Table 2. It is understood that inferring an antigenic determinant depending on the analytical criteria used, the exact position of the antigenic determinant may vary slightly.

Table 3 summarizes the expression profile of the polynucleotides corresponding to the clones disclosed in Table 1. The first column is the cd for each contig sequence disclosed in Table 1.
A specific clone identifier "Clone Number (Clone Number) for NA clones.
ID) ". The "library code" in the second column shows the expression profile of the library of tissues and / or cell lines expressing the polynucleotide of the present invention. The code for each library in the second column shows the library code provided in Table 5 and the tissue / cell source identifier code that corresponds to the library description. Expression of these polynucleotides was not observed in other tissue and / or cell libraries tested. Those of skill in the art routinely use this information to identify tissues that show a predominant expression pattern of the corresponding polynucleotides of the invention, or to predominate and / or
Alternatively, a polynucleotide that exhibits specific tissue expression can be identified.

The first column of Table 4 provides the nucleotide sequence identifier “SEQ ID NO: X”. this"
SEQ ID NO: X "matches the nucleotide sequence SEQ ID NO: X disclosed in column 5 of Table 1. The second column of Table 4 is the chromosomal arrangement of the polynucleotide corresponding to SEQ ID NO: "Cytologic Band or Chromosome".
romosome) ". The chromosome arrangement is NCBI (National
Center for Biotechnology Information
n) Determined by finding the exact match for the EST and cDNA contained in the UniGene database. Considering the putative chromosome arrangement, Online Mendelian Inheritance in M
an (Online Mendelian Inheritance in M
an, OMIM ^™ . McKusick-Nathans Institute
for Genetic Medicine, Johns Hopkins
University (Baltimore, MD) and National
Center for Biotechnology Information
, National Library of Medicine (Bethes)
Disease-related loci were determined by comparison with Morbid Map from da, MD) 2000). World Wide Web URL: http: /
/ Www. ncbi. nlm. nih. gov / omim /). When the putative chromosomal arrangement of the query overlaps with the chromosomal sequence number position of the Morbid Map entry, the OMIM reference identification number (OMIM) of the morbid map entry
(reference identification numbers) to "O
"MIM ID" and is shown in the third column of Table 4. The cues for the OMIM reference identification numbers are shown in Table 6.

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

Table 6 provides clues to the OMIM reference identification numbers disclosed in the third column of Table 4. The number of OMIM reference identifications (1st column) is Online Mendelian
Inheritance in Man (Online Mendelian)
Inheritance in Man, OMIM ^™ . McKusick-
Nathans Institute for Genetic Medici
ne, Johns Hopkins University (Baltimor)
e, MD) and National Center for Biotechn
LOGO INFORMATION, National Library o
f Medicine (Bethesda, MD) 2000) World Wi
de Web URL: http: // www. ncbi. nlm. nih. g
ov / omim /). Column 2 provides diseases associated with the cytoplasmic bands disclosed in column 2 of Table 4, as determined using the Morbid Map database.

[0090]

[Table 2]

[0091]

[Table 3]

[0092]

[Table 4]

[0093]

[Table 5]

[0094]

[Table 6] 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 produced by a combination of these methods. Polypeptides that have been prepared. Means for preparing such polypeptides are well understood in the art.

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

The polypeptides of the present invention are preferably provided in isolated form and are preferably substantially purified. Recombinantly produced versions of polypeptides (including secreted polypeptides) may be derived from techniques described herein or otherwise known in the art (eg, Smith and John).
Son, Gene 67: 31-40 (1988) by a one-step method). The polypeptide of the present invention also comprises
For example, using antibodies described herein or other techniques known in the art, such as the 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 relates to the polypeptide sequence of SEQ ID NO: Y and / or the ATCC deposit Z
There is provided a polypeptide comprising or consisting of a polypeptide encoded by the cDNA contained therein. Polynucleotide sequences comprising or consisting of the polypeptide sequence of SEQ ID NO: and / or the cDNA sequence encoded by the cDNA contained in ATCC Deposit Z are also encompassed by the invention. .

Signal Sequence The present invention also includes the mature form of the polypeptide having the polypeptide sequence of SEQ ID NO: and / or the polypeptide sequence encoded by the cDNA in the deposited clone. A polynucleotide encoding the mature form (eg, the polynucleotide sequence of SEQ ID NO: X and / or the cDNA of the deposited clone)
(Such as the polynucleotide sequences contained in) are also encompassed by the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretory leader sequence that is cleaved from the mature protein once transport of the growing protein chain across the rough endoplasmic reticulum is initiated. . Most mammalian and even insect cells cleave secreted proteins with the same specificity.
However, in some cases the cleavage of the secreted protein is not completely uniform and
This results in more than one mature species of this protein. Furthermore, it has long been known that the cleavage specificity of secreted proteins is ultimately determined by the primary structure of the intact 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 a short N-terminal charged region followed by an uncharged region of the intact (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. . The accuracy of predicting breakpoints of known mammalian secretory proteins for each of these methods is in the range of 75-80% (
von Heinje, supra). However, the two methods do not always produce 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 Signa
A computer program called IP (Henrik Nielsen et al.,
Protein Engineering 10: 1-6 (1997)), the program predicts the location of proteins in cells based on their amino acid sequences. As part of the computer prediction of this localization, McGeoc
The method of h and von Heinje is incorporated. By this program,
Analysis of the amino acid sequences of the secreted proteins described herein provided the results shown in Table 1.

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

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

(Polynucleotide and Polypeptide Variants) The present invention provides a modification of the polynucleotide sequence disclosed in SEQ ID NO: X, its complementary strand, and / or the cDNA sequence contained in the deposited clone. Regarding the body

The invention also includes 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 the polynucleotide or polypeptide of the invention but retains their essential properties. In general, variants are wholly closely similar and, in many regions, identical to the polynucleotides or polypeptides of the invention.

The present invention also provides, 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 the cDNA contained in the deposited clone, and / or a polynucleotide fragment of any of these nucleic acid molecules (eg, herein At least 80%, 85%, 90%, 95% relative to the described fragments)
, 96%, 97%, 98%, or 99% identical to a nucleic acid molecule that comprises or consists of a nucleotide sequence. Polynucleotides that hybridize to these nucleic acid molecules under stringent hybridization conditions or less stringent conditions, polypeptides encoded by these polynucleotides are also encompassed by the invention.

The invention also includes, for example, the polypeptide sequence set forth in SEQ ID NO: Y, the polypeptide sequence encoded by the cDNA contained in the deposited clone, and / or any of these polypeptides. At least 80 for polypeptide fragments (eg, fragments described herein)
%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to or consisting of an amino acid sequence.

By a nucleic acid having a nucleotide sequence which is at least 95% “identical” to a reference nucleotide sequence of the present invention, the nucleotide sequence of the nucleic acid is 100 nucleotides each of the reference nucleotide sequence, the nucleotide sequence encoding a polypeptide. It is intended to be identical to a reference sequence, except that up to 5 point mutations can be included. In other words, at least 9 relative to the reference nucleotide sequence
Up to 5% of the nucleotides of the reference sequence can be deleted or replaced by another nucleotide in order to obtain a nucleic acid having a nucleotide sequence of 5% identity,
Alternatively, a large number of nucleotides, up to 5% of the total nucleotides in the reference sequence, can be inserted in 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.

As a practical matter, any particular nucleic acid molecule or polypeptide will be at least 80%, 85%, 90%, 95%, 96%, 9% relative to the nucleotide sequence of the invention.
Whether 7%, 98%, or 99% identical can be conventionally determined using known computer programs. A preferred method for determining the best overall compatibility (also referred to as global sequence alignment) between a query sequence (sequence of the invention) and a subject sequence is described by Brutlag et al. (Comp. App
． Biosci. 6: 237-245 (1990)) based on the FASTDB computer program. In a sequence alignment, both the query and subject sequences are DNA sequences. RNA
Sequences can be compared by converting U to T. The results of this global sequence alignment are expressed as percent identity (%). Preferred parameters used in the FASTDB alignment of DNA sequences to calculate percent identity are: Matrix = Unitary, k-tuple = 4, Mismatch P.
energy = 1, Joining Penalty = 30, Randomiz
ation Group Length = 0, Cutoff Score = 1,
Gap Penalty = 5, Gap Size Penalty 0.05,
Windows 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 the 5'or 3'deletions (as opposed to due to internal deletions), manual corrections must be made to the results. This is because the FASTDB program does not consider 5'and 3'truncations of the subject sequence when calculating percent identity. For a subject sequence that is cleaved at the 5'or 3'end, the percent identity to the query sequence is the 5'and 3'of the subject sequence that is not matched / aligned as a percentage of the total bases of the query sequence. Corrected by calculating the number of bases in. Whether the nucleotides are matched / aligned is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated by the FASTDB program above using the specified parameters to arrive at the final percent identity score. This corrected score is what is used for the purposes of the present invention. Only the bases outside the 5'and 3'bases of the subject sequence that are not matched / aligned with the query sequence, as indicated by the FASTDB alignment, are calculated for the purpose of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned 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 shows no match / alignment at the first 10 bases at the 5'end. 1
0 unpaired bases represent 10% of the sequence (number of bases at the non-matching 5'and 3'ends / total number of bases in the query sequence), so 10% is FASTD
Subtracted from the percent identity score calculated by the B program.
If the remaining 90 bases were an exact match, the final percent identity would be 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 there are no bases at the 5'or 3'of the subject sequence that are not matched / aligned with the query. In this case, FASTD
The percent identity calculated by B is not manually corrected. Again, only the 5'or 3'bases of the subject sequence that do not match / align with the query sequence are manually corrected. No other manual correction is made for the purposes of the present invention.

By the polypeptide having an amino acid sequence which is at least 95% “identical” to the query amino acid sequence of the present invention, the amino acid sequence of the target polypeptide is such that the target polypeptide sequence is each of the query amino acid sequences. It is intended to be identical to the query sequence, except that changes of up to 5 amino acids per 100 amino acids may be included. In other words, up to 5% of the amino acid residues in the sequence of interest are inserted, deleted, (indels) in order to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence.
Or it may be replaced by another amino acid. These modifications of the reference sequence can occur at the amino-terminal or carboxy-terminal positions of the reference amino acid sequence, or anywhere between those terminal positions, either individually between the residues in the reference sequence or the reference sequence. Interspersed in any of one or more consecutive groups within.

As a practical matter, any particular polypeptide may be encoded, for example, by the amino acid sequence shown in Table 1 (SEQ ID NO: Y) or by 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 can be conventionally determined using known computer programs. The best overall match between the query sequence (the sequence of the invention) and the subject sequence (
The preferred method for determining (also referred to as the overall sequence alignment) is Bru
tlag et al. (Comp. App. Biosci. 6: 237-245 (1990).
)) Algorithm-based FASTDB computer program. In a sequence alignment, the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The results of the above global sequence alignments are given in percent identity. FASTD
The preferred parameters used for the B amino acid alignment are: Matrix = PAM
0, k-tuple = 2, Mismatch Penalty = 1, 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 target amino acid sequence (whichever is shorter).

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

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. Deletions occur at the N-terminus of the subject sequence, and therefore the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (mismatched N-terminus and C
Represents the number of residues at the end / total number of residues in the query sequence, and the result is FAST
10% is subtracted from the percent identity score calculated by the DB program. If the remaining 90 residues were perfectly matched, the final percent identity would be 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 residues at the N-terminus or C-terminus of the subject sequence that are not matched / aligned with the query sequence. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only residue positions outside the N- and C-termini of the subject sequence that do not match / align with the query sequence, as shown in the FASTDB alignment, are manually corrected.
No other manual correction is made for the purposes of the present invention.

Variants may include changes in the coding regions, non-coding regions, or both. Especially preferred are those that produce silent substitutions, additions or deletions,
Polynucleotide variants containing changes that do not alter the properties or activity of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Furthermore, 5 in any combination
Variants in which -10, 1-5, or 1-2 amino acids are substituted, deleted, or added are also preferred. Polynucleotide variants optimize codon expression for a variety of reasons (eg, codon expression for a particular host (codons in human mRNA are described in E.
for a preferred codon by a bacterial host such as E. coli)),
Can be generated.

Naturally occurring variants are referred to as “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., edited by John Wiley &
Sons, New York (1985)). These allelic variants can vary at either the polynucleotide level and / or the polypeptide level and are included in the 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 change the properties of the polypeptides of the invention. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function. Ron et al. Biol. Ch
em. 268: 2984-2988 (1993) authors 3, 8, or 2
A modified KGF protein was reported that had heparin binding activity even after deletion of the 7 amino terminal amino acid residues. Similarly, interferon-gamma, after deleting 8-10 amino acid residues from the carboxy terminus of this protein,
It showed higher activity up to 0-fold (Dobeli et al., J. Biotechnol.
7: 199-216 (1988)).

In addition, a wealth of evidence demonstrates that variants often retain activities 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 generate 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 every potential amino acid position. The investigators found that "the majority of molecules can be altered with little effect on either [binding activity or biological activity]." (See summary). In fact, of the over 3,500 nucleotide sequences tested,
Only 23 unique amino acid sequences produced proteins that differed significantly in activity from the wild type.

Furthermore, even if the deletion of one or more amino acids from the N-terminus or C-terminus of the polypeptide results in an alteration or deletion of one or more biological functions, other biological functions. Activity can still be retained. For example, the ability of a deletion variant to induce and / or bind an antibody that recognizes a secreted form is the ability to remove less than the majority of the secreted form from the N-terminus or C-terminus. Seems to be retained. Whether a particular polypeptide lacking the N-terminal or C-terminal residues of a protein retains such immunogenic activity depends on conventional methods described herein, and so on. If not, it can be easily determined by a conventional method known in the art.

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

The first strategy exploits the tolerance of amino acid substitutions by natural selection during the course of evolution. Conserved amino acids can be identified by comparing amino acid sequences in different species. These conserved amino acids appear to be important for protein function. In contrast, the amino acid positions at which the substitutions were tolerated by natural selection indicate that these positions are not important for protein function. Thus, the positions that tolerate amino acid substitutions may be modified, yet still maintain the biological activity of the protein.

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

As the authors mention, these two strategies have revealed that the protein is surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be tolerated at particular 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, but surface side chain characteristics are generally poorly conserved. In addition, tolerant conservative amino acid substitutions include the substitution of aliphatic or hydrophobic amino acids Ala, Val, Leu, and Ile; the substitution of Ser and Thr for hydroxyl residues; the substitution of Asp and Glu for acidic residues; Amide residue Asn
And Gln, basic residues Lys, Arg, and His; aromatic residues Phe, Tyr, and Trp, and small-sized amino acids Ala, Ser, Thr, Met, and Gly. Including replacement.

In addition to conservative amino acid substitutions, variants of the invention include (i) substitution with one or more non-conservative amino acid residues, where the amino acid residue to be replaced is by the genetic code. It may or may not be the encoded amino acid residue, 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 to increase the stability and / or solubility of the polypeptide (eg, polyethylene glycol)), or (iv) an additional amino acid (eg, IgG). Fc fusion region peptide, or fusion of the polypeptide with a leader or secretory sequence, or a sequence that facilitates purification), or (v) the polypeptide and another compound, such as albumin (but not limited to Altered albumin (eg, US Pat. No. 5,876,969 issued Mar. 2, 1999, EP Patent No. 0 413 622, and U.S. Pat. No. 5,766,666 issued Jun. 16, 1998). 883 (
These include fusions with))))), which is incorporated by reference herein in its entirety. Such variant polypeptides are considered to be within the skill of those in the art given the teachings herein.

For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged amino acids or neutral amino acids may have proteins with improved properties (eg, less aggregation). Can be generated. Aggregation of pharmaceutical formulations results in both reduced activity and increased clearance due to the immunogenic activity of the aggregate. (Pinckard 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 invention comprises at least one amino acid substitution, but 50
Amino acids of the invention having an amino acid sequence of no more than amino acid substitutions, even more preferably no more than 40 amino acid substitutions, even more preferably no more than 30 amino acid substitutions, and even more preferably no more than 20 amino acid substitutions. A polypeptide comprising a sequence. Of course, in order of increasing preference, a peptide or polypeptide comprises at least one amino acid substitution, but
It is highly preferred to have an amino acid sequence which comprises the amino acid sequence of the invention not exceeding 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. In certain embodiments, the number of additions, substitutions, and / or deletions in the amino acid sequences of the invention or fragments thereof (eg, mature forms and / or other fragments described herein) is between 1 and 5, 5-10, 5-25, 5-50, 10-50, or 50
~ 150 and conservative amino acid substitutions are preferred.

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

In the present invention, a "polynucleotide fragment" refers to a short polynucleotide having the nucleic acid sequence that is: a polynucleotide contained in the deposited clone or encoded by the cDNA in the deposited clone. Part of the nucleic acid sequence encoding the peptide; part of the nucleic acid sequence set forth in SEQ ID NO: X or their complements, or part of the polynucleotide sequence encoding the polypeptide of SEQ ID NO: Y. is there. The nucleotide fragments of the invention are 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. It has a length of 150 nt. 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 stated values (at either or both termini a few (5, 4, 3, 2, or 1) nucleotides greater or less than this). Value) is included. These nucleotide fragments have uses, including but not limited to, use as diagnostic probes and primers, as discussed herein. Of course, larger fragments (eg 50, 150, 500, 600, 2000 nucleotides)
Is preferred.

Furthermore, typical examples of the polynucleotide fragment of the present invention include, for example,
Fragments that include or consist of sequences of the following approximate number of nucleotides are included: 1-50, 51-100, 101-150, 151-20.
0, 201-250, 251-300, 301-350, 351-400, 40
1-450, 451-500, 501-550, 551-600, 651-70
0, 701-750, 751-800, 800-850, 851-900, 90
1-950, 951-1000, 1001-1050, 1051-1100, 1
101 to 1150, 1151 to 1200, 1201 to 1250, 1251 to 13
00, 1301-1350, 1351-1400, 1401-1450, 145
1-1500, 1501-1550, 155-1600, 1601-1650
, 1651 to 1700, 1701 to 1750, 1751 to 1800, 1801
1850, 1851 to 1900, 1901 to 1950, 1951 to 2000, or 2001 to the end of SEQ ID NO: X, a complementary strand thereof, or a cDNA contained in the deposited clone. In this context, "about" means
Included are ranges specifically mentioned, and ranges larger or smaller by a few nucleotides (5, 4, 3, 2, or 1) at either or both ends. Preferably, these fragments encode a polypeptide having biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides that hybridize to these nucleic acid molecules under stringent hybridization conditions or under lower stringency conditions are also included in the invention, as are the polypeptides encoded by these polynucleotides.

In the present invention, “polypeptide fragment” refers to an amino acid sequence which is a part of the amino acid sequence encoded by the cDNA contained in SEQ ID NO: Y or contained in the deposited clone. A protein (polypeptide) fragment may be "free-standing," or within a larger polypeptide (wherein the fragment may be a portion or region, most preferably as a single continuous region). Form). Representative examples of polypeptide fragments of the present invention include, for example, fragments that contain or consist of the following approximate number of amino acids: 1-20, 21-40, 41-. 60, 61-80, 81-100, 10
2-120, 121-140, 141-160, or 161 to the end of the coding region. Further, the polypeptide fragment is 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" means
Included are ranges or values specifically stated, and ranges or values that are several amino acids greater or lesser than this (5, 4, 3, 2, or 1) at either or both termini. Polynucleotides encoding these polypeptides are also included in the invention.

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

Fragments of polypeptides and polynucleotides characterized by structural or functional domains (eg, α-helix and α-helix forming regions, β-sheet and β-sheet forming regions, turns and turn forming regions) ,
Coil and coil-forming region, hydrophilic region, hydrophobic region, α-amphipathic region, β-amphipathic region, flexible region, surface-forming region, substrate-binding region, and fragment containing high antigenicity indicator region) It is also preferable. Polypeptide fragments of SEQ ID NO: Y contained within the conserved domain are specifically contemplated by the present invention. In addition, polynucleotides encoding these domains are also contemplated.

Other preferred polypeptide fragments are biologically active fragments. A biologically active fragment is a fragment that exhibits similar activity but is not necessarily identical to that of the polypeptide of the invention. The biological activity of this fragment may include improved desired activity or reduced undesired activity. Polynucleotides encoding these polypeptide fragments are also
Included in the present invention.

Preferably, the polynucleotide fragment of the present invention encodes a polypeptide that exhibits functional activity. By a polypeptide that exhibits "functional activity" is meant a polypeptide that may exhibit one or more known functional activities associated with a 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 antibodies to the polypeptide of the invention], immunogenicity ( The ability to produce antibodies that bind to the polypeptides of the invention), the ability to form multimers with the polypeptides of the invention, and the ability to bind to receptors or ligands for the polypeptides of the invention. Not limited.

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

For example, assaying for the ability of a polypeptide of the invention to bind or compete with a full-length polypeptide of the invention for binding to an antibody, in one embodiment, various immunoassays known in the art. Can be used. Such assays include radioimmunoassays, ELISAs (enzyme linked immunosorbent assays), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (eg colloidal gold, enzyme or Radioisotope labeling), western blot, precipitation reaction, agglutination assay (eg gel agglutination assay, hemagglutination assay), complement fixation assay,
Included, but 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 the identified polypeptide of the invention, or the polypeptide fragment, variant, or derivative of the invention, is assessed for its ability to multimerize, binding is, eg, , Reduced and non-reduced gel chromatography, protein affinity chromatography, and affinity blotting can be performed by means well known in the art.
Generally, Phizicky, E .; Et al., 1995, Microbiol. Rev
． 59: 94-123. In another embodiment, the physiological correlation (signal transduction) of binding of a polypeptide of the invention to its substrate can be assayed.

In addition, the assays described herein (see Examples) and other assays known in the art refer to polypeptides of the invention and fragments, variants, derivatives and analogs thereof. , Can be routinely applied to determine the ability (either in vitro or in vivo) to elicit a relevant biological activity related to that of the polypeptide of the invention. Other methods are known to those of skill in the art and are within the scope of this 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 to the polynucleotide sequence contained in ATCC Deposit No. Z, or to the complement of the sequence of SEQ ID NO: X. An epitope of the polypeptide sequence encoded by the polynucleotide, or a polynucleotide sequence contained in ATCC Deposit No. Z under stringent or lower stringency hybridization conditions as defined above. Includes polypeptides that include or consist of epitopes of the encoded polypeptide sequence. The invention further provides a polynucleotide sequence that encodes an epitope of a polypeptide sequence of the invention (eg, the sequence disclosed in SEQ ID NO: X), a polynucleotide sequence of the complementary strand of a polynucleotide sequence that encodes an epitope of the invention, And a polynucleotide sequence that hybridizes to a complementary strand under stringent hybridization conditions as defined above or under lower stringency hybridization conditions.

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

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

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

Similarly, immunogenic epitopes can be used to induce antibodies, eg, according to methods well known in the art. (For example, Sutcliffe et al. (Supra); Wils
on et al. (supra); Chow et al., Proc. Natl. Acad. Sci. USA
82: 910-914; and Bittle et al., J. Am. 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 may be presented to elicit an antibody response against an animal system (eg rabbit or mouse) with a carrier protein (eg albumin), or the polypeptide may be If sufficiently long (at least about 25 amino acids), the polypeptide can be presented without a carrier. However, immunogenic epitopes containing as few as 8-10 amino acids
It has been shown to be sufficient (e.g. in Western blotting) to raise antibodies capable of (at least) binding to the linear epitope of the denatured polypeptide.

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

As will be appreciated by those in the art, and as discussed above, the polypeptides of the present invention comprising immunogenic or antigenic epitopes can be fused to other polypeptide sequences. For example, the polypeptides of the present invention are immunoglobulin (Ig
A, IgE, IgG, IgM) constant domains or parts thereof (CH1, C)
H2, CH3, or any combination thereof and portions thereof, or albumin (recombinant albumin (eg, US Pat. No. 5,876,969 issued Mar. 2, 1999, EP 0 413 622). , And US Pat. No. 5,766,883, issued Jun. 16, 1998, which are hereby incorporated by reference in their entirety), and are not limited). Can be fused to yield a chimeric polypeptide. Such fusion proteins may facilitate purification and increase half-life in vivo. this is,
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 mammalian immunoglobulins has been shown. See, for example, EP 394,827; Traunecker et al., Nature, 331: 84-86 (1988). Enhanced delivery of antigens across the epithelial barrier to the immune system has been demonstrated for antigens bound to FcRn binding partners such as IgG or Fc fragments (eg insulin) (eg PCT Publication WO 96/22024 and The same WO99 / 0
4813). IgG fusion proteins having a disulfide-bonded dimer structure due to disulfide bonds of the IgG moiety are also more effective in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone Was found. For example, Fountoulakis et al. B
iochem. , 270: 3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (eg, a hemagglutinin (“HA”) tag or a flag tag) for detection and purification of the expressed polypeptide. Can assist. For example, Ja
The system described by nknecht et al. allows easy purification of native fusion proteins expressed in human cell lines (Janknecht et al., 1991,
Proc. Natl. Acad. Sci. USA 88: 8972-897).
In this system, the gene of interest is subcloned into a vaccinia recombination plasmid so that the open reading frame of this gene is translationally fused to an amino-terminal tag consisting of 6 histidine residues. This tag serves as the substrate binding domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2 + nitriloacetic acid-agarose columns, and histidine-tagged proteins can be selectively eluted with imidazole-containing buffer.

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 utilized to modulate the activity of the polypeptides of the invention and this method can be used to produce polypeptides with modified activity, as well as agonists and antagonists of these polypeptides. Generally, US Pat. Nos. 5,605,793; 5,811,238; 5,83.
No. 0,721; No. 5,834,252; and No. 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., J. Am. M
ol. Biol. 287: 265-76 (1999); and Lorenzo
And Blasco, Biotechniques 24 (2): 308-13.
(1998), each of which is incorporated herein by reference in its entirety. In one embodiment, modification of the polynucleotides corresponding to SEQ ID NO: X and the polypeptides encoded by these polynucleotides can be accomplished by DNA shuffling. D
NA shuffling involves assembling two or more DNA segments to produce changes in polynucleotide sequence by homologous or site-specific recombination. In another embodiment, the polynucleotide of the present invention or its encoded polypeptide is error-prone prior to recombination.
ne) It may be modified by subjecting it to random mutagenesis by PCR, random nucleotide insertion or other methods. In another embodiment, one or more components, motifs, sections, portions, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention are one or more components, motifs, of one or more heterologous molecules. , Sections, parts, domains, fragments and the like.

Antibodies Furthermore, the 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
Alternatively, an antibody that immunospecifically binds to an epitope and a T cell antigen receptor (T
CR). The antibody of the present invention is a polyclonal antibody, a monoclonal antibody,
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 against antibodies of the invention), and epitope binding fragments of any of the above, but not limited thereto. As used herein, the term "antibody" refers to
It refers to an immunologically active portion of an immunoglobulin molecule and an immunoglorin molecule, that is, a molecule containing an antigen-binding site that immunospecifically binds to an antigen. The immunoglobulin molecules of the invention can be of any type of immunoglobulin molecule (eg, IgG, IgE, I
gM, IgD, IgA, and IgY), any class (eg, IgG1, I
gG2, IgG3, IgG4, IgA1 and IgA2) or any subclass. In a preferred embodiment, the immunoglobulin molecule of the invention is IgG
It is 1. In another preferred embodiment, the immunoglobulin of the present invention is IgG4.

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

Antibodies of the invention can be monospecific, bispecific, trispecific or of greater multispecificity. A multispecific antibody may be specific for different epitopes of a polypeptide of the invention, or both a polypeptide of the invention and a heterologous epitope (eg, a heterologous polypeptide or a solid support material). Can be specific to. For example, PCT Publication WO 93/17715; WO 9
2/08802; WO 91/00360; WO 92/05793; Tu
tt et al. Immunol. 147: 60-69 (1991); U.S. Pat. No. 4
, 474,893, 4,714,681, 4,925,648,
No. 5,573,920, No. 5,601,819; Kostelny et al.
J. Immunol. 148: 1547-1553 (1992).

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

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

The invention also provides antibodies to the epitopes of the invention, as determined by any method known in the art for determining competitive binding, such as the immunoassays described herein. Antibodies that competitively inhibit the binding of In a preferred embodiment, the antibody is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% competitively bound to an epitope. Inhibit.

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

The present invention also recognizes receptor-specific antibodies that interfere with both ligand binding and receptor activation, as well as receptor-ligand complexes, and preferably specific unbound receptors or unbound ligands. It has the characteristics of a receptor-specific antibody that is not recognized by. Similarly, the present invention binds ligands and neutralizing antibodies that prevent the binding of ligands to receptors, and ligands, thereby preventing receptor activation, but not ligand binding to receptors. Contains antibodies. Further, the invention includes antibodies that activate the receptor. These antibodies may act as receptor agonists, ie enhance or activate either all or a subset of the biological activation of ligand-mediated receptor activation by, for example, inducing receptor dimerization. obtain. The antibody may be specified as an agonist, antagonist or inverse agonist for a biological activity, including the specific biological activity of the peptides of the invention disclosed herein. The antibody agonist can be produced using a method known in the art. For example, PCT Publication WO 96/40281; US Pat. No. 5,811,097; Deng et al., Blood 92 (6): 1981-19.
88 (1998); Chen et al., Cancer Res. 58 (16): 366
8-3678 (1998); Harrop et al., J. Am. Immunol. 161 (4
): 1786-1794 (1998); Zhu et al., Cancer Res: 58.
(15): 3209-3214 (1998); Yoon et al., J. Am. Immunol
． 160 (7): 3170-3179 (1998); Prat et al. Cell
． Sci. 111 (Pt2): 237-247 (1998); Pittard et al.,
J. Immunol. Methods 205 (2): 177-190 (199).
7); Liautard 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 entireties.

Antibodies of the invention can be used, for example, but not limited to, to purify, detect, and target the polypeptides of the invention. These include both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibody has use in immunoassays for qualitatively and quantitatively measuring the levels of polypeptides of the invention in biological samples. For example, Harl
ow et al., Antibodies: A Laboratory Manual, (
Cold 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 invention can be used either alone or in combination with other compounds. The antibody can be further recombinantly fused at the N-terminus or the C-terminus to a heterologous polypeptide, or can be chemically linked (including covalent and non-covalent) to the polypeptide or other composition. . For example, the antibodies of the present invention can be recombinantly fused or conjugated to effector molecules such as molecules and heterologous polypeptides, agents, radionuclides, or toxins that are useful as labels in detection assays. For example, PCT publication WO92 / 084
95; WO91 / 14438; WO89 / 12624; US Pat. No. 5,314.
995; and EP 396,387.

The antibodies of the invention are modified (ie, covalently attached).
attachment), by covalent attachment of any type of molecule to the antibody such that the antibody does not prevent it from producing an anti-idiotypic response. For example, without limitation, antibody derivatives include, for example, glycosylations, acetylations, pegylations, phosphorylations.
n), amidation, known blocking / blocking groups
Antibodies modified by derivatization with, proteolytic cleavage, ligation to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more non-classical amino acids.

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

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

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art, and are discussed in detail in the Examples (eg, Example 16). In a non-limiting example, mice can be immunized with a polypeptide of the invention or cells expressing such a peptide. Once an immune response is detected (eg, antibody specific for the antigen is detected in mouse serum)
The mouse spleen is harvested and splenocytes 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. The hybridoma clones are then assayed for cells secreting antibodies capable of binding the polypeptides of the invention by methods known in the art. Ascites fluid, which generally contains high levels of antibodies
) Can be produced by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides a method 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, this antibody A hybridoma is a hybridoma clone that fuses myeloma cells with splenocytes isolated from a mouse immunized with the antigen of the present invention and then secretes an antibody capable of binding to the polypeptide of the present invention.
Produced by screening the hybridomas resulting from the fusion.

Antibody fragments that recognize particular epitopes can be generated by known techniques. For example, Fab and F (ab ′) 2 fragments of the invention are (Fa
It can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce the b fragment) or pepsin (to produce the F (ab ') 2 fragment). F (ab ′) 2 fragments contain 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 which carry the polynucleotide sequences encoding them. In certain embodiments, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (eg, human or mouse). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with the antigen (eg, using labeled antibody or antigen bound or captured to a solid surface or bead). Phage used in these methods are typically phage having the antibody domain of Fab, Fv or disulfide-stabilized Fv recombinantly fused to either the phage gene III protein or the phage gene VIII protein. Is a filamentous phage containing the fd and M13 binding domains expressed from E. coli.
Examples of phage display methods that can be used to generate the antibodies of the invention include those disclosed below: Brinkman et al. Immu
nol. Methods 182: 41-50 (1995); Ames et al., J. Am.
Immunol. Methods 184: 177-186 (1995); Ke.
tttleborough et al., Eur. J. Immunol. 24: 952-95
8 (1994); Persic et al., Gene 187 9-18 (1997);
Burton et al., Advances in Immunology 57:19.
1-280 (1994); PCT application number PCT / GB91 / 01134; PC
T publication WO 90/02809; WO 91/10737; WO 92/010
47; WO 92/18619; WO 93/11236; WO 95/159.
82; WO 95/20401; and US Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717.
No. 5,427,908; No. 5,750,753; No. 5,821.
No. 047; No. 5,571, 698; No. 5,427, 908; No. 5,5.
No. 16,637; No. 5,780,225; No. 5,658,727; No. 5,733,743 and No. 5,969,108 (each of which is herein incorporated by reference). Incorporated as a reference in its entirety).

As described in the above references, after phage selection, the region encoding the antibody derived from the phage may be cloned to produce whole antibodies, including human antibodies, or any other desired antigen binding fragment. It can be separated and used and 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 with methods known in the art, such methods being disclosed below. R: PC
T Publication WO 92/22324; Mullinax et al., BioTechniq.
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 may 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 parts of the antibody are derived from different animal species (for example, 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.
hnies 4: 214 (1986); Gillies et al., (1989) J.
． Immunol. Methods 125: 191-202; US Pat. No. 5,
807,715; 4,816,567; and 4,816397, which are hereby incorporated by reference in their entireties.
. 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 the non-human species and a framework region from a human immunoglobulin molecule. . Often, framework residues within the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter (preferably improve) antigen binding. Substitutions of these frameworks are identified by methods well known in the art, for example modeling CDR and framework residue interactions to identify framework residues important for antigen binding, and at specific positions. By sequence comparison to identify unusual framework residues. (For example, Queen et al., US Pat. No. 5,585,0.
89; Riechmann et al., Nature 332: 323 (1988), which are incorporated herein by reference in their entirety. )
Antibodies can be humanized using a variety of techniques known in the art including: For example, CDR-grafting (European Patent 239,400; PCT Publication WO 91/09).
967; U.S. Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (European Patent 592,106). No. 519,596; Padlan, Molecular Immunology.
28 (4/5): 489-498 (1991); Studnicka et al., P.
protein engineering 7 (6): 805-814 (1994)
); Roguska et al., PNAS 91: 969-973 (1994)), and chain shuffling (US Pat. No. 5).
, 565, 332).

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

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins but which are capable of expressing human immunoglobulin genes. For example, a complex of human heavy and light chain immunoglobulin genes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions and diversity regions (diversity)
region) can be introduced into mouse embryonic stem cells in addition to the human heavy chain gene and human light chain gene. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletions in the JH region interfere with endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the normal fashion with a selected antigen (eg, all or part of a polypeptide of the invention). Monoclonal antibodies directed against the antigen can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgene carried in the transgenic mouse is B
It reconstitutes during cell differentiation and then undergoes class switching and somatic mutations. 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 technique for producing human antibodies and human monoclonal antibodies, as well as protocols for producing such antibodies, see, eg, PCT Publications WO98 / 24893; WO92 / 01047; WO96 / 340.
96; WO96 / 33735; European Patent No. 0 598 877; US Patent No. 5,
No. 413,923; No. 5,625,126; No. 5,633,425; No. 5,569,825; No. 5,661,016; No. 5,545,806.
No. 5,814,318; No. 5,885,793; No. 5,916.
771; and 5,939,598, which are hereby incorporated by reference in their entireties. In addition, Abgenix, Inc. (F
Companies such as Reemont, CA) and Genpharm (San Jose, CA) may be engaged in providing human antibodies to selected antigens using techniques similar to those described above.

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

Furthermore, antibodies against the polypeptides of the present invention may in turn be utilized to produce anti-idiotypic antibodies that "mimic" the polypeptides of the present 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). ) For example, binding and multimerization of polypeptides.
cation) and / or an antibody that competitively inhibits binding of the polypeptide of the invention to a ligand is used to produce an anti-idiotype that "mimics" the multimerization domain and / or binding domain of the polypeptide. Obtain and, as a result, bind and neutralize the polypeptide and / or its ligand.
Neutralization of such anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligands. For example, such anti-idiotypic antibodies can be used to bind to a polypeptide of the invention and / or to bind its ligand / receptor, thereby blocking its biological activity.

Polynucleotides Encoding Antibodies The present invention further provides polynucleotides comprising nucleotide sequences encoding the antibodies of the present invention and fragments thereof. The invention also provides an antibody (eg, as defined above) under stringent or less stringent hybridization conditions, preferably which specifically binds to a polypeptide of the invention, preferably , An antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO: Y).

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

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

Once the nucleotide sequence of the antibody and the corresponding amino acid sequence are determined,
Nucleotide sequences of antibodies may be prepared using methods well known in the art for manipulation of 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., 1998, Current Protocols.
in Molecular Biology, John Wiley & Sons
, NY. Both are incorporated herein by reference in their entirety. )) Can be engineered to produce antibodies with different amino acid sequences, eg, to make amino acid substitutions, deletions, and / or insertions.

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

Further, by splicing a gene derived from a mouse antibody molecule of appropriate antigen specificity with a gene derived from a human antibody molecule 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)) may be used. As mentioned above, a chimeric antibody is a molecule in which different portions are derived from different animal species, and such molecules have variable regions derived from the constant regions of mouse mAb and human immunoglobulin (eg, humanized antibody). .

Alternatively, described techniques for the production of single chain antibodies (US Pat. No. 4,946,46)
778; Bird, Science 242: 423-42 (1988); H.
uston et al., Proc. Natl. Acad. Sci. USA 85: 587.
9-5883 (1988); and Ward et al., Nature 334: 544.
-54 (1989)) can be adapted to the production of single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region through amino acid bridges, yielding single chain polypeptides. E. 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 of Producing Antibodies The antibodies of the invention may be produced by any method known in the art for synthesizing antibodies, in particular by chemical synthesis or, preferably, by recombinant expression techniques. obtain.

Antibodies of the invention, or fragments, derivatives or analogs 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 the polynucleotide encoding the antibody molecule of the invention or the heavy or light chain of the antibody, or a portion thereof (preferably containing the variable domain of the heavy or light chain) is obtained, production of the antibody molecule Vectors for can be 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 to construct expression vectors containing antibody coding 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 invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. I will provide a.
Such a vector may be a constant region of an antibody molecule (eg PCT Publication WO86 /
05807; PCT Publication WO 89/01036; and US Pat. No. 5,122.
, 464), and the variable domain of the antibody can be cloned into such a vector for total expression of heavy or light chains.

The expression vector is transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibody of the invention. Accordingly, the invention includes a host cell containing 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 double-chain antibodies, the vectors encoding both heavy and light chains are co-expressed in a host cell for expression of entire immunoglobulin molecules, as detailed below. Can be done.

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

In bacterial systems, many expression vectors may be advantageously selected depending on the use intended for the expression of the antibody molecule. For example, if large quantities of such proteins are to be produced, a vector that directs high levels of expression of the easily purified fusion protein product may be desired for the production of pharmaceutical compositions of antibody molecules. Such vectors include, but are not limited to: The antibody-encoding sequence, in frame with the lacZ-encoding region, in the vector.
e) can be separately ligated, so that a fusion protein is produced in E. 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 .; Biol. Chem. 24: 5503-5509
(1989)) and so on. The pGEX vector can also be used to express foreign polypeptides as a fusion protein with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily 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 contain 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 to express heterologous genes. This virus grows in Spodoptera frugiperda cells. The antibody coding sequences can be individually cloned into non-essential regions of the virus (eg the polyhedrin gene) and placed under control of the AcNPV promoter (eg the polyhedrin promoter).

In mammalian host cells, a number of virus-based expression systems may be utilized. When an adenovirus is used as an expression vector, the sequence encoding the antibody of interest may be linked to the 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 nonessential regions of the viral genome (eg, E1 or E3 regions) result in recombinant viruses that are viable and capable of expressing antibody molecules in infected hosts (eg, Logan & Shenk).
, Proc. Natl. Acad. Sci. USA 81: 355-359 (1
984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals are ATG
It includes a start codon and adjacent sequences. In addition, this initiation codon ensures that translation of the entire insert is ensured by the reading frame of the desired coding sequence (
must be in phase with the 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 enhanced 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 of protein products (
Cleavage, for example) can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells can be used that have the cellular machinery for the precise processing of primary transcription, glycosylation, and phosphorylation of the gene product. Such mammalian host cells include CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, WI38, and especially, for example, BT48
3, breast cancer cell lines such as Hs578T, HTB2, BT20 and T47D, and normal breast cell lines such as, for example, CRL7030 and Hs578Bst.

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 containing a viral origin of replication, the host cell may choose suitable expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.).
, And a DNA controlled by a selectable marker. Heterogeneous DN
Following the introduction of A, the engineered cells can be grown in enriched medium for 1-2 days,
Then, the medium is switched to the selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, and the cell stably integrates the plasmid into its chromosome and proliferates to form cell foci, which in turn can be cloned and propagated. Can become a cell line. This method can be advantageously used to engineer cell lines that express antibody molecules. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

A number of selection systems may be used, including herpes simplex virus thymidine kinase (Wigler et al., Cell 11: 223 (1977)), hypoxanthine-guanine phosphoribosyl transferase (Szybalska & S.
zybalski, Proc. Natl. Acad. Sci. USA 48: 2
02 (1992)), and adenine phosphoribosyl transferase (L
owy et al., Cell 22: 817 (1980)), but without limitation, these genes can be used in tk-, hgprt- or aprt- cells, respectively. Antimetabolite resistance can also be used as a basis for selection of the following genes: dhfr, which 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 the aminoglycoside G-.
Conferring resistance to 418 (Clinical Pharmacy 12: 4)
88-505; Wu and Wu, Biotherapy 3: 87-95 (
1991); Tolstoshev, Ann. Rev. Pharmacol. T
oxicol. 32: 573-596 (1993); Mulligan, Sci.
ence 260: 926-932 (1993); and Morgan and.
Anderson, Ann. Rev. Biochem. 62: 191-217
(1993); May 1993, TIB TECH 11 (5): 155-21.
5); and hygro, which confers resistance to hygromycin (
Santerre et al., Gene 30: 147 (1984)). Methods well known in the field of recombinant DNA technology can be routinely applied to select the desired recombinant clones, and such methods are described below: eg Ausu.
bel et al. (ed.), Current Protocols in Molecule
ar Biology, John Wiley & Sons, NY (1993);
Kriegler, Gene Transfer and Express
n, A Laboratory Manual, Stockton Press
, NY (1990); and Chapters 12 and 13, Dracopoli et al. (Eds.), Current Protocols in Human Genetics.
s, John Wiley & Sons, NY (1994); Colberre-
Garapin et al. Mol. Biol. 150: 1 (1981), which are incorporated herein by reference in their entirety.

Expression levels of antibody molecules can be increased by vector amplification (for review,
Bebington and Hentschel, The use of ve
ctors based on gene amplification fo
r the expression of cloned genes in
mammalian cells in DNA cloning, Vol. Three
． (See Academic Press, New York, 1987). When 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 amplified region is linked to the antibody gene, antibody production is also increased (Crouse et al., Mol. Cell. Biol. 3: 257.
(1983)).

A host cell may be co-transfected with two expression vectors of the invention, a first vector encoding a polypeptide from the heavy chain and a second vector encoding a polypeptide from the light chain. . The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used, which encodes and expresses both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid excess toxic free heavy chain (Pr
audfoot, Nature 322: 52 (1986); Kohler, P.
roc. Natl. Acad. Sci. USA 77: 2197 (1980)).
. The coding sequences for the heavy and light chains may include cDNA or genomic DNA.

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

The present invention provides a polypeptide of the invention (or a portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 of this polypeptide,
Or 100 amino acids), 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 is a polypeptide of the invention (or a portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70,
(80, 90, or 100 amino acids). For example, by fusing or conjugating a polypeptide of the invention to an antibody specific for a particular cell surface receptor, either in vitro or in vivo, the polypeptide of the invention is directed against a particular cell type. To target
Antibodies can be used. Antibodies fused or conjugated to the polypeptides of the invention can also be used in in vitro immunoassays and purification methods using methods known in the art. See, eg, Harbor et al., Supra, and PCT Publication WO93.
/ 21232; EP 439,095; Naramura et al., Immunol. L
ett. 39: 91-99 (1994); US Patent No. 5,474,981; G.
illies et al., PNAS 89: 1428-1432 (1992); Fell.
Et al., J. Immunol. 146: 2446-2452 (1991). These are incorporated by reference in their entirety.

The invention further includes compositions comprising a polypeptide of the invention fused or linked to a domain other than the variable region of an antibody. For example, the polypeptides of the invention can be fused or conjugated to the Fc region of an antibody, or a portion thereof. The part 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 whole domains or portions thereof. These polypeptides can also be fused or conjugated to portions of the above antibodies to form multimers. For example, an Fc portion fused to a polypeptide of the invention can form a dimer through a disulfide bond between the Fc portions. Higher multimeric forms can be made by fusing the polypeptide to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the invention to antibody moieties are known in the art. For example, US Pat. Nos. 5,336,603; 5,622,929; 5,359,04.
No. 6, No. 5,349,053; No. 5,447,851; No. 5,112.
, 946; EP 307,434; EP 367,166; PCT publication WO9.
6/04388; WO91 / 06570; Ashkenazi et al., Proc. N
atl. Acad. Sci. USA 88: 10535-10539 (1991).
); Zheng et al. Immunol. 154: 5590-5600 (199
5); and Vil et al., Proc. Natl. Acad. Sci. USA 89
: 11337-11341 (1992) (the above references are incorporated by reference in their entireties).

As discussed above, the polypeptide, polypeptide fragment, or polypeptide corresponding to a variant of SEQ ID NO: is provided to increase the in vivo half-life of the polypeptide, or It can be fused or conjugated to the antibody moieties described above for use in immunological assays using methods known in the art. further,
The polypeptide corresponding to SEQ ID NO: Y is fused or linked to the above antibody moiety,
Purification can be facilitated. One reported example describes 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 mammalian immunoglobulins (EP 394). , 8
27; Traunecker et al., Nature 331: 84-86 (1988).
)). The polypeptides of the invention, fused or linked to an antibody having a disulfide-linked dimeric structure (due to IgG), also bind to other molecules than monomeric secreted proteins or protein fragments alone and It may be more efficient at neutralizing (Funtoulakis et al., J. Biochem. 270).
: 3958-3964 (1995)). In many cases, the Fc portion of fusion proteins may be beneficial in therapy and diagnosis, thus resulting, 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 may interfere with therapy and diagnosis. For example, in drug discovery, h
Human proteins such as IL-5 have been fused to the Fc portion for the purpose of high throughput screening assays to identify antagonists of hIL-5 (Bennett et al., J. Molecular Recognitio.
n 8: 52-58 (1995); Johnson et al., J. Am. Biol. Che
m. 270: 9459-9471 (1995)).

In addition, the antibodies of the present invention or fragments thereof can be fused to a marker sequence, such as a peptide that facilitates purification. In a preferred embodiment, the marker amino acid sequence is inter alia a hexa-histidine peptide (eg pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chats.
The tag, provided by W. Worth, CA, 91311), and many of these marker amino acid sequences are commercially available. For example, Gentz et al., Proc
． Natl. Acad. Sci. Hexa-histidine provides convenient purification of fusion proteins, as described in USA 86: 821-824 (1989). Another peptide tag useful for purification is the "HA" tag (Wilson et al., Ce, which corresponds to an epitope derived from influenza hemagglutinin protein.
ll37: 767 (1984)), and "flag" tags.

The 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 substances, luminescent substances, bioluminescent substances, radioactive substances, positron emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions. , Can be mentioned. This detectable substance is an antibody (or fragment thereof)
, Either directly or indirectly, via a mediator using techniques known in the art, such as a linker known in the art, or linked. See, eg, 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 include biotin; 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.

In addition, the antibody or fragment thereof may be used in therapeutic moieties (eg cytotoxins (eg cytostatic or cytotoxic agents)), therapeutic agents or radioactive metal ions (eg α-emitters (eg 213Bi)). Etc.). Cytotoxic or cytotoxic agents include any agent that is harmful to cells. Examples include paclitaxol, cytochalasin B, gramicidin D,
Ethidium bromide, emetine, mitomycin, etoposide, teniposide (ten
oposide), vincristine, vinblastine, colchicine (colch)
icin), doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol,
And puromycin, and analogs or homologues thereof. The therapeutic agents include antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine (5-f
luourouracyl decarbazine), an alkylating agent (eg,
Mechlorethamine, thioepa chlorambucil, melphalan,
Carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracycline (eg, daunorubicin (eg, daunorubicin). Formerly daunomycin) and doxorubicin), antibiotics such as dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents such as vincristine and vinblastine. ), But
It is not limited to them.

The conjugates of the invention can be used to modify a given biological response and the therapeutic agent or drug moiety is not construed as limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide that has the desired biological activity. Such proteins include, for example, toxins (eg, abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin); proteins (eg,
Tumor necrosis factor, a-interferon, β-interferon, nerve growth factor,
Platelet-derived growth factor, tissue plasminogen activator, apoptotic drug (eg, TNF-α, TNF-β, AIM I (International Publication No. WO97 / 33899).
No.), AIM II (see WO 97/34911), Fas ligand (Takahashi et al., Int. Immunol. 6).
: 1567-1574 (1994)), VEGI (International Publication No. WO99 / 231).
05)), thrombotic or anti-angiogenic agents (eg, angiostatin or endostatin); or biological response modifiers (eg, lymphokines, interleukin-1 (“IL-1”)). , Interleukin-2 (“IL
-2 "), interleukin-6 (" IL-6 "), granulocyte macrophage colony stimulating factor (" GM-CSF "), granulocyte colony stimulating factor (" G-CSF ").
), Or other growth factors, etc.).

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

Techniques for coupling such therapeutic moieties to the antibody are well known, eg Arnon.
Et al., "Monoclonal Antibodies For Immuno.
targeting Of Drugs In Cancer Therapy "
Monoclonal Antibodies And Cancer The
rapid, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Lis.
s, Inc. 1985); Hellstrom et al., "Antibodies F."
or Drug Delivery "Controlled Drug Del
Ivery (2nd edition), Robinson et al. (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
applications, Pinchera et al. (eds.), pp. 475-506 (198).
5); "Analysis, Results, And Future Pros
selective Of The Therapeutic Use Of Ra
dilabelled Antibody In Cancer Therap
y ”Monoclonal Antibodies For Cancer D
inspection And Therapy, Baldwin et al. (eds.), 303
-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Prop.
parties Of Antibody-Toxin Conjugates "
, Immunol. Rev. 62: 119-58 (1982).

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

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

These techniques apply to hematological malignancies (ie, minimal residual disease (MRD) in patients with acute leukemia).
And "Nonself" in Transplantation to Prevent Graft-versus-Host Disease (GVHD)
Allows the screening of specific populations of cells as they can be found with the cells. Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells that may undergo proliferation and / or differentiation as may be found in human cord blood.

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

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

Western blot analysis generally involves the steps of preparing a protein sample, electrophoresing the protein sample on a polyacrylamide gel (eg 8% -20% SDS-PAGE depending on the molecular weight of the antigen), polyacrylamide the protein sample. Transfer from gel to membrane (eg nitrocellulose, PVDF or nylon), blocking membrane in blocking solution (eg PBS with 3% BSA or non-fat milk) washing membrane (eg PBS) -Tween 20), blocking the membrane with a primary antibody (antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, diluting with blocking buffer The enzyme A secondary antibody (eg 32P or 125I) conjugated to a substrate (eg horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (eg 32P or 125I)
This involves blocking the membrane with a primary antibody (eg recognizing an anti-human antibody), washing the membrane in wash buffer, and detecting the presence of antigen. Those skilled in the art are familiar with parameters that can be modified to increase the signal detected and reduce background noise. For further discussion on Western blot protocols, see, eg, Aus.
ubel et al. (eds.), 1994, Current Protocols in M.
molecular Biology, Vol. 1. John Wiley & So
ns, Inc. , New York (10.8.1).

ELISA involves the steps of preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, binding a detectable compound such as an enzyme substrate (eg horseradish peroxidase or alkaline phosphatase). The antibody of interest is added to the well and incubated for a period of time, and the presence of antigen is detected. In the ELISA, the antibody of interest need not be bound to the detectable compound; instead, a second antibody (which recognizes the antibody of interest) bound to the detectable compound can be added to the wells. Further, instead of coating the well with the antigen, the antibody can be coated to the well. In this case, a second antibody conjugated to a detectable compound can be added subsequent to the addition of the antigen of interest to the coated wells. One of ordinary skill in the art will be aware of parameters that can be modified to increase the signal detected, and other variations of the ELISA known in the art. For further discussion on ELISA, see, eg, Ausubel et al., Ed., 1994, Cur.
rent Protocols in Molecular Biology,
Volume 1, John Wiley & Sons, Inc. , New York,
See 11.2.1.

Antibody binding affinity for antigen and off-rate of antibody-antigen interaction (of
f-rate) can be determined by competitive binding assay. 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. Detection of antibody bound to the antigen. The affinity of the antibody of interest for a particular antigen, and the binding off-rate can be determined from the data by Scatchard plot analysis. Competition with the second antibody can also be determined using a radioimmunoassay. In this case, the antigen is the labeled compound (eg 3H or 125I) in the presence of increasing amounts of unlabeled secondary antibody.
A) antibody of interest conjugated to

Therapeutic Uses The present invention further relates to antibody-based therapies, which comprise treating an antibody of the invention to treat one or more of the disclosed diseases, disorders, or conditions in animals. , Preferably a mammal, and most preferably a human patient. The therapeutic compounds of the invention include the antibodies of the invention (including fragments, analogs and derivatives thereof, as described herein) and nucleic acids encoding the antibodies of the invention (described herein). Including fragments, analogs and derivatives thereof and anti-idiotypic antibodies). Antibodies of the invention include diseases, disorders or conditions associated with aberrant expression and / or activity of the polypeptides of the invention (including any one or more of the diseases, disorders, or conditions described herein). (But not limited to) can be used to treat, inhibit or prevent. Treatment and / or prevention of diseases, disorders or conditions associated with aberrant expression and / or activity of the polypeptides of the invention includes, but is not limited to, ameliorating the symptoms associated with those diseases, disorders or conditions. Not done.
The antibodies of the present invention are known in the art or can be provided in a pharmaceutically acceptable composition as described herein.

One summary of how the antibodies of the invention may be used therapeutically is locally or systemically in the body or (eg, by complement (CDC) or by effector cells (ADCC)). The direct cytotoxicity of the antibody (as mediated)
Conjugating a polynucleotide or polypeptide of the invention. Some of these approaches are described in more detail below. Given the teachings provided herein, one of ordinary skill in the art would be able to determine, without undue experimentation, how to use the antibodies of the invention for diagnostic, monitoring or therapeutic purposes. Recognize.

The antibodies of the invention may be other monoclonal or chimeric antibodies, or lymphokines or hematopoietic growth factors (eg, IL-2, IL-2, IL-3 and IL
-7) and the like) can be advantageously used.

The antibodies of the invention can be administered alone or in combination with other types of treatments such as radiation therapy, chemotherapy, hormone therapy, immunotherapy and antitumor agents.
Generally, it is preferred to administer a species-sourced or species-reactive product that is the same species as the patient's species (in the case of antibodies). Thus, in a preferred embodiment, human antibodies,
The fragment derivative, analog, or nucleic acid is administered to a human patient for treatment or prevention.

[0213]   Polynucleotides or polypeptides of the invention, including fragments thereof
) Immunoassays, and for the treatment of disorders associated therewith
, With high affinity and / or affinity for a polypeptide or polynucleotide of the invention.
Or potent in vivo inhibitory and / or neutralizing antibodies, their fragmentation
It is preferable to use the component, or the region thereof. Such an antibody, Fragmen
Or region is preferably a polynucleotide or polypeptide of the invention.
It has an affinity for (including fragments thereof). Preferred binding affinity
As 5 x 10^-2M, 10^-2M, 5 x 10^-3M, 10^-3M, 5 x 1
0^-4M, 10^-4M, 5 x 10^-5M, 10^-5M, 5 x 10^-6M, 10^{− 6} M, 5 x 10^-7M, 10^-7M, 5 x 10^-8M, 10^-8M, 5 x 10^{− 9} M, 10^-9M, 5 x 10^-10M, 10^-10M, 5 x 10^-11M, 10 ^-11 M, 5 x 10^-12M, 10^-12M, 5 x 10^-13M, 10^-13M
5 x 10^-14M, 10^-14M, 5 x 10^-15M, and 10^-15M
And binding affinities with smaller dissociation constants or Kd's.

Gene Therapy In certain embodiments, a nucleic acid comprising a sequence encoding an antibody or functional derivative thereof treats a disease or disorder associated with aberrant expression and / or activity of a polypeptide of the invention, Administered for the purpose of gene therapy to inhibit or prevent. Gene therapy refers to a therapy that is performed by administering 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.

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

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

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

Delivery of the nucleic acid to the 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 transfected with the nucleic acid in vitro). Converted and then transplanted to the patient). These two approaches are known as in vivo gene therapy or as ex vivo gene therapy, respectively.

In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where they are expressed to produce the encoded product. This may be done by any of a number of methods known in the art (eg by constructing them as part of a suitable nucleic acid expression vector and administering it intracellularly (eg defective Sexually or attenuated retrovirus or other viral vectors (see US Pat. No. 4,980,286), or
Liposomes, by direct injection of naked DNA, or by the use of microparticle bombardments (eg, gene gun; Biolistic, Dupont), or by coating with lipids or cell surface receptors, or by transfecting drugs. It is bound to a ligand that undergoes receptor-mediated endocytosis by encapsulation in microparticles or microcapsules, or by administering them in association with peptides known to enter the nucleus. By administration (eg Wu and Wu, J. et al.
Biol. Chem. 262: 4429-4432 (1987)).
(Which can be used to target cell types that specifically express the receptor). In another embodiment, a nucleic acid-ligand complex may be formed where the ligand disrupts endosomes fusogenic.
) Contains viral peptides to ensure that the nucleic acid avoids lysosomal degradation. In yet another embodiment, the nucleic acids can be targeted in vivo for cell-specific uptake and expression by targeting specific receptors (eg, PCT Publication No. WO92 / 06180; WO92 /). No. 22635; No. WO92 / 20316; No. WO93 / 14188, No. WO93 / 202.
21). Alternatively, the nucleic acid can be introduced intracellularly and incorporated into host cell DNA for expression by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86.
: 8932-8935 (1989); Zijlstra et al., Nature 34.
2: 435-438 (1989)).

In a specific embodiment, viral vectors that contain nucleic acid sequences that encode the antibodies of the invention are used. For example, a retroviral vector 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. The nucleic acid sequence encoding the antibody used in gene therapy is cloned into one or more vectors, which facilitates delivery of the gene into patients. For more details on retroviral vectors, see Boesen et al., Bio.
therapy 6: 291-302 (1994), which describes the use of retroviral vectors to deliver the mdrI gene to hematopoietic stem cells to make 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); S.
almons and Gunzberg, Human Gene Therapy
4: 129-141 (1993); and Grossman and Wils.
on, Curr. Opin. in Genetics and Devel. Three
: 110-114 (1993).

Adenovirus is another viral vector that can be used in gene therapy. Adenoviruses are particularly attractive vehicles for delivering genes to respiratory epithelia. Adenovirus naturally infects respiratory epithelia where it causes 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, Current Opin
Ion in Genetics and Development 3:49
9-503 (1993) provides an overview of adenovirus-based gene therapy.
Bout et al., Human Gene Therapy 5: 3-10 (1994).
) Demonstrated the use of an adenovirus vector to transfer a gene to the airway epithelium of rhesus monkeys. Another example of the use of adenoviruses in gene therapy is Ro
senfeld et al., Science 252: 431-434 (1991); R.
osenfeld et al., Cell 68: 143-155 (1992); Mast.
rangeli et al. Clin. Invest. 91: 225-234 (19
93); PCT Publication No. WO 94/12649; and Wang et al., Gene.
Therapy 2: 775-783 (1995). In a preferred embodiment, adenovirus vectors are used.

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

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

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

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

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

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

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

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

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

Therapeutic / Prophylactic Administration and Compositions The present invention provides for treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. To provide a method. 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 such as but not limited to cows, pigs, horses, chickens, cats, dogs, and the like, and preferably mammals, and most preferably humans.

The formulations and methods of administration that can be used when the compound comprises nucleic acids or immunoglobulins are described above; additional suitable formulations and routes of administration are among those described herein below. Can be selected from

Various delivery systems are known and can be used to administer the compounds of the invention (eg, liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing the compounds, Receptor-mediated endocytosis (eg, Wu and Wu, J. Biol. Chem. 262: 4429-4432 (
1987)), the 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 skin linings, such as the oral, rectal and intestinal mucosa, and others. Can be administered with any of the biologically active agents of Administration can be systemic or local. Further, the pharmaceutical compounds or compositions of the invention may be administered by any suitable route, including intraventricular and intrathecal injections; intraventricular injection may be, for example, an intraventricular catheter attached to a reservoir, such as an Ommaya reservoir. Can be facilitated by) to introduce into the central nervous system. Pulmonary administration may also be employed, eg, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In certain embodiments, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this is for example, but not limited to, surgery. By local injection, topical application (eg in combination with post-surgical wound dressing), injection, by catheter, by suppository, or by an implant (the implant being a membrane such as a sialastic membrane or Porous, non-porous, or gelatinous materials, including fibers). Preferably, when administering a protein of the invention, including an antibody,
Care must be taken to use materials where proteins are not absorbed.

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

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., N .; Engl. J. Med. 321
574 (1989)). In another embodiment, polymeric materials may be used (Medical Applications of Control).
led Release, Langer and Wise (ed.), CRC Pre
s. , Boca Raton, Florida (1974); Control
ed Drug Bioavailability, Drug Product
Design and Performance, Smolen and Bal
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., J. Am. Neurosurg. 71: 105 (
1989) also). In yet another embodiment, the controlled release system can be placed near the therapeutic target, ie, the brain, thus requiring only a portion of the systemic dose (eg, Goodson, Medical Applicat).
ions of Controlled Release, (supra), Volume 2,
115-138 (1984)).

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

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

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 in the United States Pharmacopeia or other Means that it is listed in the generally accepted pharmacopoeia.
The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers include sterile liquids such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin (eg, peanut oil, soybean oil, mineral oil, sesame oil, etc.). 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 used 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 can, if desired, also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. 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 E.I. W. "Remin" by Martin
gton's Pharmaceutical Sciences ". Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition can also include a solubilizer and a local anesthesia such as lignocaine to relieve pain at the site of injection. Generally, the components are either separately or mixed together in a single dosage form, eg, in ampoules or sachets (s) showing a fixed amount of active agent.
supplied as a lyophilized powder or water-free concentrate in a sealed container such as an achette). Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, the ingredients may be mixed prior to administration,
Ampoules 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 salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid and the like, and sodium, potassium, ammonium, calcium, ferric hydroxide, Isopropylamine, triethylamine,
Mention may be made of salts formed with cations such as those derived from 2-ethylaminoethanol, histidine, procaine and the like.

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

For antibodies, the dosage administered to a patient is typically 0.1 mg to 100 mg per kg patient body weight. Preferably, the dosage administered to a patient is between 0.1 mg and 20 mg / kg of the patient's body weight, more preferably 1 mg to 10 mg / kg of the patient's body weight. In general, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, low dosages and infrequent administrations of human antibodies are often possible. Further, the dosage and frequency of administration of the antibodies of the invention may be altered (eg,
It can be reduced by enhancing antibody uptake by lipidation and the like 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 ingredients of the pharmaceutical compositions of the invention. Notifications in the form prescribed by governmental agencies regulating the manufacture, use or sale of pharmaceutical or biological products may optionally be accompanied by such containers. This notice
It reflects the agency's approval of manufacture, use or sale for human administration.

Diagnostic and Imaging Labeled antibodies that specifically bind to the polypeptide of interest, and their derivatives and analogs thereof, have been used for diagnostic purposes to detect abnormal expression and expression of the polypeptides of the invention. Diseases, disorders and / or conditions associated with activity may be detected, diagnosed or monitored. The present invention provides for the detection of aberrant expression of a polypeptide of interest, which comprises (a) using one or more antibodies specific for the polypeptide of interest to target the protein in an individual's cells or body fluids. Assaying the expression of the polypeptide of
And (b) comparing the level of gene expression to the level of standard gene expression, whereby increasing or decreasing the level of assayed polypeptide gene expression compared to the standard level of expression. Shows abnormal expression.

The present invention provides a diagnostic assay for diagnosing a disorder, which assay comprises (
a) assaying the expression of the polypeptide of interest in cells or fluids of an individual using one or more antibodies specific for the polypeptide of interest, and (b) this gene expression level and standard Comparing the level of gene expression, whereby an increase or decrease in the assayed polypeptide gene expression level compared to its standard expression level is indicative of a particular disorder. With respect to cancer, the presence of relatively high amounts of transcripts in biopsied tissue from an individual may indicate a predisposition to the development of the disease, or provide a means for detecting the disease prior to the onset of actual clinical symptoms. You can A more definitive diagnosis of this type may allow health professionals to use preventative measures or earlier aggressive treatment, thereby preventing the development or further progression of cancer.

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

One aspect of the present invention is the detection and diagnosis of diseases or disorders associated with aberrant expression of the polypeptide of interest in animals, preferably mammals, and most preferably humans. In one embodiment, diagnosis is a) administering to a subject (eg, parenterally, subcutaneously, or intraperitoneally) an effective amount of a labeled molecule that specifically binds to a 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 the background level; and d) detecting the labeled molecule in the subject, so that the label is above this background level. Detection of the activating molecule indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by a variety of methods, including comparing the amount of labeled molecule detected to 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 needed to produce a diagnostic image. In the case of a radioisotope moiety, the amount of radioactivity injected for a human subject will usually be about 5
It is in the range of 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the particular protein.
In vivo tumor imaging is described by S. W. Burchiel et al., "Immunophar
macokinetics of Radiolabeled Antibod
ies and Their Fragments "(Tumor Image
ng: The Radiochemical Detection of Ca
ncer, Chapter 13, S. W. Burchiel and B.I. A. Rhodes, Masson Publishing Inc. (1982).

[0250] 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 will not be bound. The time interval after administration is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours, which allows the C. to be cleared to background levels.
In another embodiment, the post-administration time interval 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 the first diagnosis, 6 months after the first diagnosis, the first diagnosis). 1 year later).

The presence of labeled molecule can be detected from the patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. One of ordinary 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 present invention include, but are not limited to, computer tomography (CT), whole body scanning such as position emission tomography (PET), magnetic resonance imaging (MRI). ), And ultrasonography.

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

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

In another particular embodiment of the invention, the kit is a diagnostic kit for use in screening sera containing antibodies specific for proliferative and / or cancerous polynucleotides and polypeptides. is there. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may comprise a substantially isolated polypeptide antigen containing the epitope. This epitope specifically immunoreacts with at least one anti-polypeptide antigen antibody. Further, such a kit comprises means for detecting the binding of the above antibody to an antigen (eg, the antibody is a fluorescent compound (eg, fluorescein or rhodamine that can be detected by flow cytometry) and a conjugate). Can be). In certain embodiments, the kit 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.

[0256] In a more specific embodiment, the detection means of the kit described above comprises a solid support to which the polypeptide antigens described above are attached. Such a kit may 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 described above.

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

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

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

The invention therefore provides an assay system or kit for carrying out this diagnostic method. The kit generally comprises a support having surface-bound recombinant antigens,
And a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

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

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

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

Further, the polypeptides (including fragments, and especially epitopes) of the present invention include immunoglobulin (IgA, IgE, IgG, IgM) constant domains or portions thereof (CH1, CH2, CH3, and any thereof). Combination of (
(Including both whole domains and parts thereof)) to give chimeric polypeptides. These fusion proteins facilitate purification and exhibit increased half-life in vivo. One reported example is human CD4
A chimeric protein consisting of the first two domains of the polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins has been described (EP A 394,827; Traunecker et al. Nature 3).
31: 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 secretory proteins or protein fragments alone (Funtoulakis et al. , J. Biochem. 270: 3.
958-3964 (1995)). Polynucleotides comprising, or composed of, nucleic acids encoding these fusion proteins are also encompassed by the invention.

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869).
No.) discloses fusion proteins comprising various parts of the constant regions of immunoglobulin molecules together with another human protein or parts thereof. In many cases, the Fc portion of fusion proteins may be beneficial in therapy and diagnosis, thus resulting, 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 may interfere with therapy and diagnosis. For example, in drug discovery, human proteins such as hIL-5 have been fused to Fc moieties for the purpose of high throughput screening assays to identify antagonists of hIL-5 (D. Bennett et al. J. Molecular Reco
gnition 8: 52-58 (1995); Johnson et al.
Biol. Chem. 270: 9459-9471 (1995)).
.

In addition, the polypeptides of the present invention can be fused to a marker sequence (eg, a peptide that facilitates purification of the fused polypeptide). In a preferred embodiment, the marker amino acid sequence is inter alia a hexa-histidine peptide (eg p
QE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, CA, 91311)),
Many of these marker amino acid sequences are commercially available. For example, Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1
989), hexa-histidine provides convenient purification of the fusion protein. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein (Wi.
Lson et al., Cell 37: 767 (1984)).

Thus, any of these above fusions can be engineered using the polynucleotides or polypeptides of the invention. Vectors, Host Cells, and Protein Production The present invention also relates to vectors containing the polynucleotides of the present invention, host cells, and the production of polypeptides by recombinant techniques. For example, the vector can be a phage vector, a plasmid vector, a viral vector, or a retroviral vector. Retroviral vectors can be replication competent or replication defective. In the latter case, viral propagation generally will be complete (complementary).
only occurs in host cells.

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

The polynucleotide insert may be labeled with a suitable promoter (for example, the phage λPL promoter, the E. coli lac promoter, the trp promoter, the phoA promoter and the tac promoter, the SV40 early promoter and the SV40 late promoter, to name a few). Should be operably linked to the LTR promoter). Other suitable promoters are known to those of skill in the art. The expression construct further comprises sites for transcription initiation, transcription termination,
And in the transcribed region, it contains a ribosome binding site for translation. The coding portion of the transcript expressed by the construct is preferably a translation initiation codon initially, and a termination codon (UA) appropriately located at the end of the polypeptide to be translated.
A, UGA or UAG).

As indicated, the expression vector preferably contains at least one selectable marker. Such markers include dihydrofolate reductase, G418, or neomycin resistance genes for eukaryotic cell culture, as well as E. coli. It contains a tetracycline, kanamycin or ampicillin resistance gene for cultivation in E. coli and other bacteria. Representative examples of suitable hosts are bacterial cells (eg E. c.
oli, Streptomyces and Salmonella typhim
urium cells); fungal cells such as yeast cells (eg, Saccharomy)
ces cerevisiae or Pichia pastoris (ATC
C Accession No. 201178)); Drosophilia S2 and Spodo
Insect cells such as ptera Sf9 cells; CHO cells, COS cells, 293 cells, and animal cells such as Bowes melanoma cells; and plant cells, but are not limited thereto. Appropriate culture media and conditions for the above-described host cells are known in the art.

Among the preferred vectors for use in bacteria are pQE70, pQE6.
0 and pQE-9 (available from QIAGEN, Inc.); pBluesc
ript vector, Phagescript 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 the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (S
(available from tratagene); and pSVK3, pBPV, pMSG
And pSVL (available from Pharmacia). Preferred expression vectors for use in yeast systems are pYES2, pYD1, pTEF1 / Z
eo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalph, pP
IC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K
, PPIC9K, and PAO815 (all Invitrogen, Carl
(available from bad, CA), but is not limited thereto. Other suitable vectors will be readily apparent to those of skill in the art.

Introduction of the construct into the host cell is carried out by calcium phosphate transfection, DE
It can be provided by AE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described by 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 lacking the recombinant vector.

Polypeptides of the invention may be ammonium sulfate precipitated or ethanol precipitated, acid extracted, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectins. It can be recovered from recombinant cell culture and purified by well known methods, including chromatography. Most preferably high performance liquid chromatography (“HPLC”)
Is used for purification.

The polypeptides of the invention, and preferably secreted forms, may also be recovered from: purified from natural sources, including body fluids, tissues and cells, whether directly isolated or cultured. A product of a chemical synthesis procedure; and
Products recombinantly produced 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 in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or non-glycosylated. Furthermore, the polypeptides of the present invention may also, in some cases, include an initial modified methionine residue as a result of host-mediated processes. Therefore, 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 post-translational protein in all eukaryotic cells. N-terminal methionine is also effectively removed in most prokaryotes in most proteins, but for some proteins this prokaryotic removal process depends on the nature of the amino acids to which the N-terminal methionine is covalently attached. And is inefficient.

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

In one example, the plasmid vector pPIC9K expresses a DNA encoding a polypeptide of the invention in a Pichea yeast system essentially as described below, as described herein. Used to: "Pic
hia Protocols: Methods in Molecular B
iology ", D.I. R. Higgins and J.M. Edited by Cregg, The H
umana Press, Totowa, NJ, 1998. This expression vector is derived from the PSF-2 of the present invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretion signal peptide (ie, leader peptide) located upstream of the multiple cloning site.
Allows expression and secretion of proteins.

[0277] Many other yeast vectors allow for the proposed expression constructs to have appropriately positioned signals for transcription, translation, secretion (if desired), etc., as will be readily appreciated by those of skill in the art. PPIC9 as long as it provides an in-frame AUG if needed)
K (for example, pYES2, pYD1, pTEF1 / Zeo, pYES2 / GS,
pPICZ, pGAPZ, pGAPZα, pPIC9, pPIC3.5, pHI
L-D2, pHIL-S1, pPIC3.5K, and PAO815).

In another embodiment, high level expression of a heterologous coding sequence (eg, such as a polynucleotide of the invention) is achieved by expressing a heterologous polynucleotide of the invention such as an expression vector (eg, pGAPZ or pGAPZα). A) and the yeast culture is grown in the absence of methanol.

In addition to including host cells containing the vector constructs discussed herein, the present invention also includes primar (primarily mammalian) vertebrate sources.
y) Includes host cells, secondary host cells, and immortalized host cells. These host cells have been engineered to delete or replace endogenous genetic material (eg, coding sequences) and / or genetic material 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 regulatory regions (eg, promoters and / or enhancers) and endogenous polynucleotide sequences via homologous recombination, such that Form a new transfer unit (eg, US Pat. No. 5,641,670 issued June 24, 1997; US Pat. No. 5,73 issued March 31, 1998).
No. 3,761; International Publication No. WO 96/29 published on September 26, 1996.
No. 411; International Publication No. WO 94/12650 published on August 4, 1994.
Koller et al., Proc. Natl. Acad. Sci. USA 86: 8
932-8935 (1989); and Zijlstra et al., Nature 3
42: 435-438 (1989). Each of these disclosures is incorporated by reference in their entirety).

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
Tur., 310: 105-111 (1984)). For example, a polypeptide corresponding to a fragment of the polypeptide sequence of the present invention can be synthesized by using a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, the common amino acid D isomers, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4
-Aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline , Homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,
Phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids (eg, b-methyl amino acids), Ca-methyl amino acids, Na-methyl amino acids, and common amino acid analogs. Furthermore, the amino acid is D
It may be (dextrorotatory) or L (levorotatory).

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

Further post-translational modifications encompassed by the invention include, for example: N-linked or O-linked carbohydrate chains (N-terminal or C-terminal processing), chemical moieties to the amino acid backbone. Attachment, chemical modification of 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 such as an enzymatic label, a fluorescent label, an isotope label or an affinity label to allow detection and isolation of the protein.

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

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

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

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 numerous coupling methods available to those of skill in the art (eg, EP 0 401 384 (G-CSF, incorporated herein by reference).
To PEG), Malik et al., Exp. Hematol. 20: 102
8-1035 (1992) (tresyl chloride)
Report also the PEGylation of GM-CSF with). For example, polyethylene glycol can be covalently attached to an amino acid residue via a reactive group, such as a free amino or carboxyl group. Reactive groups are groups to which activated polyethylene glycol molecules can be attached. The amino acid residue having a free amino group may include a lysine residue and an N-terminal amino acid residue; the amino acid residue having a free carboxyl group may include an aspartic acid residue, a glutamic acid residue and a C-terminal amino acid residue. Residues may be mentioned. Sulfhydryl residues are also
It can be used as a reactive group for attaching polyethylene glycol molecules. Preferred for therapeutic purposes is a bond at the amino group, eg at the N-terminus or at the lysine group.

As alluded to above, polyethylene glycol can be attached to proteins via attachment to any of a number of amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine residues, histidine residues, aspartic acid residues, glutamic acid residues, or cysteine residues.
Using one or more reaction chemistries, a particular amino acid residue of a protein (eg, lysine, histidine, aspartic acid, glutamic acid or cysteine) or more than one type of amino acid residue of a protein (eg, lysine, Polyethylene glycol may be attached to histidine, aspartic acid, glutamic acid, cysteine and combinations thereof.

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

As noted above, PEGylation of the proteins of the invention can be accomplished by any number of means.
Can be achieved. For example, polyethylene glycol can be attached to the protein either directly or by an intervening linker. A linkerless system for connecting polyethylene glycol to proteins is described in Delgado et al., Crit. Rev. Thera. Drug Ca
rrir Sys. 9: 249-304 (1992); Francis et al., In.
tern. J. of Hematol. 68: 1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO95 / 060.
58; and WO 98/32466. The disclosure of each of these is incorporated herein by reference.

[0290]   Polyethylene can be incorporated directly into protein amino acid residues without intervening linkers.
One system for linking ren glycol is tresylated.
MPEG (this is tresyl chloride (ClSO _Two CH_TwoCF_Three) Using monomethoxypolyethylene
(Produced by modifying Lenglycol (MPEG)).
In the reaction of protein with tresylated MPEG, polyethylene glycol
, Are directly bound to the amine groups of proteins. Therefore, the present invention relates to the tamper of the present invention.
Quality and 2,2,2-trifluoroethane
By reacting with a polyethylene glycol molecule containing a sulfonyl group,
The resulting protein-polyethylene glycol conjugate is included.

Polyethylene glycol can also be attached to proteins using many different intervening linkers. For example, US Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for attaching polyethylene glycol to proteins. Protein-polyethylene glycol conjugates, in which polyethylene glycol is attached to the protein by a linker, are also MPEG-succinimidyl succinates.
imidylsuccinate), 1,1′-carbonyldiimidazole activated MPEG, MPEG-2,4,5-trichlorophenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives Can be produced by reaction of the protein with a compound such as Many more polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO98 / 32466, the entire disclosure of which is incorporated herein by reference. PEGylated protein products produced using the reaction chemistries set forth herein are included within the scope of the invention.

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

Polypeptides of the invention include monomers or multimers (ie, dimers,
Trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomeric and multimeric polypeptides of the present invention, their preparation and compositions comprising them, preferably Therapeutics. In certain embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In a further embodiment, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

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

As used herein, the term heteromer refers to a multimer that comprises one or more heterologous polypeptides (ie, polypeptides of different proteins) in addition to the polypeptide of the present invention. In a particular embodiment, the multimers of the invention are heterodimers, heterotrimers or heterotetramers. In a further embodiment, the heteromeric multimers of the invention are at least heterodimers, at least heterotrimers or at least heterotetramers.

The multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and / or covalent attachment and / or may be indirectly linked, eg by liposome formation. Thus, in one embodiment, multimers of the invention (eg, homodimers or homotrimers) are formed when the polypeptides of the invention contact each other in solution. In another embodiment, the heteromultimer 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. It In another embodiment, the multimers of the invention are with a polypeptide of the invention, and / or
Alternatively, it is formed by a covalent bond between the polypeptides of the present invention. Such covalent bonds include one or more amino acids contained in the polypeptide sequence (eg, included in the polypeptide encoded by the polypeptide described in the Sequence Listing or encoded by the deposited clone). It may include residues. In one example, the covalent bond is a bridge between cysteine residues located within the interacting polypeptide sequence in the native (ie, naturally occurring) polypeptide. In another example, this covalent bond is the result of chemical or recombinant manipulation. Alternatively, such a covalent bond may comprise one or more amino acid residues contained in the heterologous polypeptide sequence in the fusion protein of the invention.

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

Another method for preparing the multimeric polypeptides of the invention involves the use of the polypeptides of the invention fused to a leucine zipper polypeptide sequence or an isoleucine zipper polypeptide sequence. Leucine zipper domains and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. The leucine zipper originally had some DN
Identified in A-binding proteins (Landschulz et al., Science)
240: 1759, (1988)), and since then in a variety of different proteins. Particularly known leucine zippers are naturally occurring peptides and their derivatives that form dimers or trimers. Examples of leucine zipper domains suitable for producing the soluble multimeric proteins of the present invention include PCT application WO 94/10, incorporated herein by reference.
The leucine zipper domain described at 308. Recombinant fusion proteins comprising a polypeptide of the invention fused to a dimerizing or trimerizing peptide sequence in solution are expressed in a suitable host cell and the resulting soluble multimeric fusion proteins are prepared in the art. Recovered from the culture supernatant using known techniques.

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

In another example, a protein of the invention is bound by an interaction between Flag® polypeptide sequences that are included in a fusion protein of the invention that includes a Flag® polypeptide sequence. In a further embodiment, the binding of the protein of the invention binds by interaction between the heterologous polypeptide sequence contained in the Flag® fusion protein of the invention and the anti-Flag® antibody.

The multimers of the present invention can be produced using chemical techniques known in the art.
For example, the polypeptides desired to be included in the multimers of the present invention can be chemically crosslinked using linker molecules and linker molecule length optimization techniques known in the art (eg, US Pat. , 478,925, which is incorporated herein by reference in its entirety). In addition, the multimers of the invention are produced using techniques known in the art to generate one or more cysteine residues between cysteine residues located within the sequence of the polypeptide desired to be included in the multimer. Intermolecular bridges can be formed (see, eg, US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety). further,
The polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide, and techniques known in the art have been modified with one or more of these modifications. It can be applied to generate multimers containing polypeptides (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 component desired to be included in the multimers of the invention (eg, US Pat. No. 5,4,4).
78,925, which is incorporated herein by reference in its entirety).

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

Uses of Polynucleotides Each of the polynucleotides identified herein can be used as reagents in a number of ways. The following description should be considered exemplary and utilizes known techniques.

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

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

[0306] Similarly, somatic cell hybrids carry the polynucleotide P to a particular chromosome.
It provides a rapid method for CR mapping. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotide can be accomplished using a panel of specific chromosomal fragments. Other gene mapping strategies that may be used are in situ hybridization, labeled flow sorted.
pre-screening on the flow sorted) chromosome, chromosome-specific cd
Preselection by hybridization to construct an NA library, and computer mapping techniques (eg, Shuller, Trends Biotechnol 16:45, incorporated herein by reference in its entirety).
6-459 (1998)).

The exact chromosomal location of the polynucleotide is also determined by the metaphase chromosomal spread (spr
ead) 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
See 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 panels (to mark multiple sites and / or multiple chromosomes). Can be done.

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

Once 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 coinheritance between the chromosomal location and the presentation of a particular disease. (Disease mapping data is described in, for example, V. McKusick, Mendel.
ian Inheritance in Man (Johns Hopkins
(Available online through the University Welch Medical Library)). Given 1 megabase mapping resolution and 1 gene per 20 kb, the cDNA precisely localized to the chromosomal region associated with disease could be one of 50-500 potential causative genes.

Thus, once co-inheritance is established, differences in the polynucleotides and corresponding genes between affected and unaffected individuals can be tested. First, visible structural changes in the chromosome (eg, deletions or translocations) are examined in chromosome spreads or by PCR. In the absence of structural changes, the presence of point mutations is confirmed. Mutations observed in some or all affected individuals but not in normal individuals indicate that this mutation may cause the disease.
However, complete sequencing of polypeptides and the corresponding genes from some normal individuals is required to distinguish mutations from polymorphisms. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.

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

Accordingly, the invention also provides a diagnostic method useful during the diagnosis of disorders, which method comprises the step of measuring the expression level of a polynucleotide of the invention in cells or body fluids from an individual, and Comprising a step of 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 a standard is indicative of a disorder.

[0314] In yet another embodiment, the invention includes a kit for analyzing a sample for the presence of proliferative and / or cancerous polynucleotides from a test subject. In a general embodiment, the kit comprises at least one polynucleotide probe containing 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 comprises a 31 'mer end internal to this region. Has one chain. In a further embodiment, this 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 an indicator of prognosis, and patients exhibiting enhanced or suppressed polynucleotide expression of the present invention are Experience poor clinical outcome compared to patients expressing this gene at levels closer to standard levels.

“Measuring the expression level of the polynucleotide of the present invention” means that the level of the polypeptide of the present invention or the mRNA encoding the polypeptide is directly measured in the first biological sample. Either objective (eg, by determining or assessing absolute protein or mRNA levels) or relative (eg, by comparing to polypeptide or mRNA levels in a second biological sample). It is intended to be measured or evaluated qualitatively or quantitatively. Preferably, the polypeptide level or mRNA level in the first biological sample is measured or evaluated and compared to a standard polypeptide level or mRNA level, the standard being from an unimpaired individual. Determined by averaging the levels obtained from the second biological sample obtained or from a population of unimpaired individuals. As will be appreciated in the art, once standard polypeptide or mRNA levels are known, this can be used repeatedly as a standard for comparison.

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

The methods provided above may preferably be applied to diagnostic methods and / or kits in which the polynucleotides and / or polypeptides are bound to a solid support. In one exemplary method, the support is described in US Pat. Nos. 5,837,832, 5,874,219 and 5,856,174, "
It can be a "gene chip" or a "biological chip". Further, such gene chips to which the polynucleotides of the invention are attached can be used to identify polymorphisms between this polynucleotide sequence and polynucleotides isolated from the test subject. Knowledge of such polymorphisms (ie their location as well as their presence) is valuable in identifying disease loci for many disorders, including cancerous diseases and conditions. Such methods are described in US Pat. Nos. 5,858,659 and 5,856,104. The United States patents referenced above are incorporated herein by reference in their entireties.

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

The present invention is useful for the detection of cancer in mammals. In particular, the invention is
Useful during 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); and chronic myelogenous leukemia (including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc.). Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Especially preferred is human.

Pathological cell proliferative diseases, disorders and / or conditions are often associated with inappropriate activation of protooncogenes (Gelmann, EP et al., “The Eti.
LOGO OF ACUTE LEUKEMIA: MOLECULAR GE
NETICS AND VIRAL ONCOLOGY ", Neoplasti
c Diseases of the Blood, Volume 1, Wiernik,
P. H. Et al., 161-182 (1985)). Neoplasia is now from qualitative alterations of the normal cellular gene product, by the insertion of viral sequences into the chromosome, chromosomal translocations of genes into more actively transcribed regions, or by some other mechanism, or of gene expression. It is believed to result from quantitative modification (Gelmann et al., Supra). Mutated or altered expression of certain genes appears to be involved in the pathogenesis of some leukemias, among other tissues and other cell types (Gelmann et al., Supra).
In fact, the human counterparts of some animal neoplastic oncogenes have been amplified or translocated in some cases of human leukemia and carcinoma (Gelman).
n et al., supra). For example, c-myc expression is associated with the non-lymphocytic leukemia cell line HL-6.
Highly amplified at 0. The levels of c-myc are found to be down-regulated when HL-60 cells are chemically induced to stop amplification (International Publication No. WO 91/15580). However, c-myc or c-
Exposure of HL-60 cells to a DNA construct complementary to the 5'end of myb c
-Myc protein or c-myb protein has been shown to block translation of the corresponding mRNAs that downregulate expression, resulting in arrest of cell proliferation and differentiation of treated cells (WO 91/15580). ;
Wickstrom et al., Proc. Natl. Acad. Sci. 85: 102
8 (1988); Anfossi et al., Proc. Natl. Acad. Sci.
86: 3379 (1989)). However, one of ordinary skill in the art will find the utility of the present invention in view of the large number of cells and cell types of various origins known to exhibit a proliferative phenotype, proliferative diseases, disorders of hematopoietic cells and tissues. And / or recognize that treatment of the condition is not limited.

In addition to the above, the polynucleotide may be triple helix formed or antisense DN.
It can be used to control gene expression through A or RNA. Antisense technology is described in, for example, Okano, J. et al. Neurochem. 56: 560 (1
991), "Oligodeoxynucleotides as Antis.
Sense Inhibitors of Gene Expression ",
CRC Press, Boca Raton, FL (1988). Triple helix formation is described, for example, in 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 RNA. For these techniques, the preferred polynucleotides are usually oligonucleotides 20-40 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 blockage of RNA transcription from DNA, whereas antisense RNA hybridization blocks translation of mRNA molecules into polypeptides. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an attempt to treat or prevent disease. .

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

Polynucleotides are also useful for identifying an individual from a small biological sample. 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 genomic DNA is digested with one or more restriction enzymes and probed for Southern blots to generate unique bands to identify staff. This method does not suffer from the current limitations of the "Dog tag," which can be difficult to positively identify by being lost, exchanged, 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 selected portions of the genome of an individual. These sequences can be used to prepare PCR primers to amplify and isolate such selected DNA, which can then be sequenced. Individuals can be identified using this technique because each individual has a unique set of DNA sequences. Once a unique ID database is established for an individual, whether that individual is alive or dead, positive identification of that individual can be made from a very small tissue sample.

Forensic biology will 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, lung sp (pulmonary sp)
DNA) obtained from a very small biological sample, such as a urium) or a surfactant, urine, fecal material, etc.) can be amplified using PCR.
In one 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.
an and Co. (1992)). Once these specific polymorphic loci are 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 the source of a particular tissue. Such a need arises in forensic medicine, for example, if tissue of unknown origin is provided. Suitable reagents may include, for example, DNA probes or primers specific to a particular tissue prepared from the sequences of the invention. Such a panel of reagents may identify tissues by species and / or organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contaminants.

At a minimum, the polynucleotides of the present invention “subtract from known sequences in the process of discovering new polynucleotides, as molecular weight markers in Southern gels, as diagnostic probes for the presence of specific mRNAs in specific cell types. As a probe for "selecting and making oligomers for attachment to a" gene chip "or other support, for raising anti-DNA antibodies using DNA immunization techniques, and for eliciting an immune response. Can be used as

Uses of Polypeptides Each of the polypeptides identified herein can be used in a number of ways. The following description should be considered exemplary and utilizes known techniques.

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

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

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 needed to produce a diagnostic image. In the case of a radioisotope moiety,
For human subjects, the amount of radioactivity injected is usually about 5-20 mTc.
It is in the millimeter range. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the particular protein. In vivo tumor imaging is described by S. W. Burchiel et al., “Immunopharmacokine
tics of Radiolabeled Antibodies and
Their Fragments. "(Tumor Imaging: The
The first of Radiochemical Detection of Cancer
Chapter 3, S. W. Burchiel and B.I. A. Rhodes, Masson
Publishing Inc. (1982)).

Accordingly, the invention provides a method of diagnosing a disorder, which method comprises (a) assaying the expression of a polypeptide of the invention in cells or body fluids of an individual; (b) the level of expression of this gene. Is compared 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 a disorder. For cancer, may the presence of relatively high abundance of transcripts in biopsied tissue from an individual predispose to the development of this disease?
Alternatively, it may provide a means to detect this disease prior to the onset of actual clinical symptoms.
A more definitive diagnosis of this type may allow health professionals to use earlier preventative measures or aggressive treatments, thereby preventing the onset or further progression of cancer.

In addition, the polypeptides of the present invention may be used to treat, prevent and / or diagnose disease. For example, in a patient, replacing the absence or reduced level of a polypeptide (eg, insulin) with a different polypeptide (eg, hemoglobin B).
Absent or reduced levels of hemoglobin S, SOD, catalase, DNA repair proteins) against, inhibiting the activity of the polypeptide (eg, oncogene or tumor suppressor), the activity of the polypeptide (eg, Activating (by binding to a receptor), decreasing the activity of a membrane bound receptor by competing with the membrane bound receptor for free ligands (eg soluble TNF receptors used in reducing inflammation), or Polypeptides of the invention can be administered in addressing to produce a desired response (eg, inhibition of blood vessel growth, enhancement of immune response to proliferating cells or tissues).

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

At least the polypeptides of the invention are SDS-P using methods well known to those of skill in the art.
It can be used as a molecular weight marker in 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 method of assessing transformation of host cells. 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 or preventing disorders, diseases, and conditions. This gene therapy method uses nucleic acid (DNA, RNA, and antisense DNA or RNA to achieve expression of the polypeptide of the present invention.
A.) Introducing the sequence into an animal. This method requires a polynucleotide encoding a polypeptide of the invention operably linked to a promoter and any other genetic element required for expression of this polypeptide by the target tissue. Techniques for such gene therapy and delivery are known in the art, see, eg, WO 90/11092, incorporated herein by reference.

Thus, for example, a cell derived from a patient may have a polynucleotide (DNA or R) containing a promoter operably linked to a polynucleotide of the invention ex vivo.
NA) and the engineered cells are then provided to the patient to be treated with the polypeptide. Such methods are well known in the art.
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.
erase 7: 1-10 (1996); Santodonato et al., Gene.
See Therapy 4: 1246-1255 (1997); and Zhang et al., Cancer Gene Therapy 3: 31-38 (1996). These documents are incorporated herein by reference. In one embodiment, the engineered cells are arterial cells. The arterial cells can be reintroduced into the patient by direct injection into the arteries, tissues surrounding the arteries, or by catheter injection.

As discussed in more detail below, the polynucleotide constructs may be delivered by any method that delivers an injectable substance to cells of an animal, such as tissue (heart, muscle, skin,
Injection into the interstitial space of the lungs, liver, etc.). The polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

[0340] In one embodiment, the polynucleotide of the present invention is delivered as a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to any delivery vehicle (that acts to assist, facilitate or facilitate entry into cells.
(Including viral sequences, viral particles, liposomal formulations, lipofectin, precipitants, etc.). However, the polynucleotides of the invention can also be delivered in liposomal formulations, and lipofectin formulations and the like can be prepared by methods well known to those of skill in the art. Such methods are described, for example, in US Pat. Nos. 5,593,972 and 5,589, incorporated herein by reference.
, 466 and 5,580,859.

The polynucleotide vector constructs of the invention used in the method of gene therapy are preferably not integrated into the host genome and do not contain sequences that allow 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 those of skill in the art.

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

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

The polynucleotide construct of the present invention can be used for tissue (animal, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, and Includes intestines, testes, ovaries, uterus, rectum, nervous system, eyes, glands, and connective tissue)
Can be delivered to the interstitial space of the. This interstitial space is the space between the reticulated fibers of organ tissue,
Liquid mucopolysaccharide matrix between cells, elastic fibers in the walls of blood vessels or chambers, collagen fibers of fibrotic tissue, or connective tissue sheath muscle cells (connective cells).
Includes the same matrix within the issue enheasing muscle cell) or in the lacunae of the bone. This is likewise the space occupied by circulating plasma and lymphatic fluid of the lymphatic channels. Delivery of muscle tissue to the interstitial space
Preferred for reasons discussed below. The polynucleotide construct of the present invention comprises
It can be conveniently delivered by injection into the tissue containing these cells. The polynucleotide constructs of the present invention are preferably delivered to and expressed in differentiated, persistent, non-dividing cells, which delivery and expression may be in undifferentiated cells or cells that are not fully differentiated ( (Eg, blood stem cells or dermal fibroblasts). Muscle cells in vivo take up a polynucleotide,
And it is particularly qualified in its ability to develop.

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

The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, including, for example, inhalation of aerosol formulations, especially for delivery to lung or bronchial tissues, throat or mucous membranes of the nose, among others. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in this procedure.

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

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

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

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

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. DOTAP (1,2-bis (oleoyloxy)-
For a description of the synthesis of 3- (trimethylammonio) propane) liposomes, see, eg, PCT Publication No. WO 90/11092, 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. USA, 84: 7413-7417. Similar methods can be used to prepare liposomes from other cationic lipid substances.

Similarly, anionic and neutral liposomes can be prepared, for example, by Avant.
It is readily available from i Polar Lipids (Birmingham, Ala.) or can be easily prepared using readily available materials.
Such substances include, among others, phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC).
), Dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE). These substances also
It can be mixed with DOTMA starting material and DOTAP starting material in suitable proportions. Methods of using these materials to form liposomes are well known in the art.

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

The liposomes can include multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs), with SUVs being preferred. Various liposome-nucleic acid complexes are prepared using methods well known in the art. For example, Straubinge, which is incorporated herein by reference.
r et al., Methods 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 and then hydrating with a solution of the substance to be encapsulated. SUVs are prepared by long-term sonication of MLVs, producing a homogeneous population of unilamellar vesicles. The substance to be encapsulated is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, dry lipid membranes should be treated with sterile water or 10
Resuspend in a suitable solution, such as an isotonic buffer solution such as mM Tris / NaCl, sonicate, then mix the preformed liposomes directly with the DNA. Due to the binding of the positively charged liposomes to the cationic DNA, the liposomes and DNA form a very stable complex. SUVs find use with small nucleic acid fragments. LUVs are prepared by many methods well known in the art. A commonly used method is Ca ²⁺ -EDTA chelation (Pa
pahadjopoulos et al., Biochim. Biophys. Acta,
394: 483 (1975); Wilson et al., Cell, 17:77 (197).
9)); ether injection (Deamer et al., Biochim. Biophys. A).
cta, 443: 629 (1976); Ostro et al., Biochem. Bio
phys. Res. Commum. 76: 836 (1977); Fraley.
Et al., Proc. Natl. Acad. Sci. USA, 76: 3348 (197).
9)); Surfactant dialysis (Enoch et al., Proc. Natl. Acad. Sc.
i. USA, 76: 145 (1979)); and reverse phase evaporation (RE).
V) (Fraley et al., J. Biol. Chem., 255: 10431 (19).
80); Szoka et al., Proc. Natl. Acad. Sci. USA, 75
145 (1978); Schaefer-Ridder 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 about 5: 1 to about 1: 5. More preferably, the ratio is from about 3: 1 to about 1: 3. Even more preferably, the ratio is about 1: 1.

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

In a particular embodiment, an RN comprising a sequence encoding a polypeptide of the invention.
The retroviral particles containing A are used to engineer cells ex vivo or in vivo. Examples of the retrovirus capable of inducing a retrovirus plasmid vector include Moloney murine leukemia virus, splenic necrosis virus, Rous sarcoma virus,
Harvey sarcoma virus, chicken leukemia virus, gibbon leukemia virus,
Human immunodeficiency virus, myeloproliferative sarcoma virus, and breast cancer virus are included, but are not limited to.

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

This producer cell line produces infectious retroviral vector particles which 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 cell expresses a polypeptide of the invention.

In certain other embodiments, the polynucleotides of the invention contained in adenovirus vectors are used to engineer cells ex vivo or in vivo. Adenovirus can be engineered so that it encodes and expresses the polypeptide of the 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 integration of the viral DNA into the host cell chromosome, resulting in less worry about insertional mutagenesis. Furthermore, adenovirus has been used for many years as a live vaccine for the intestine with excellent safety aspects (Schwartzet et al., Am. Re.
v. Respir. Dis. , 109: 233-238 (1974)). Finally, adenovirus-mediated gene transfer has been demonstrated in many cases, including the transfer of α-1-antitrypsin and CFTR into the lungs of cotton rats (Ro.
senfeld et al., Science, 252: 431-434 (1991); R.
osenfeld et al., Cell 68: 143-155 (1992)). Moreover, extensive research seeking to establish adenovirus as the causative agent in human cancers was uniformly negative (Green et al. Proc. Natl. Acad. S).
ci. USA, 76: 6606 (1979)).

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

Preferably the adenovirus used in the present invention is replication defective. Replication-defective adenoviruses require the help of helper viruses and / or packaging cell lines to form infectious particles. The resulting virus is capable of infecting cells and can express the polynucleotide of interest operably linked to a promoter, but is unable to replicate in most cells. A replication-defective adenovirus may be deleted at one or more of the following genes: E1a, E1b, E3, E4, E2a or all or part of L1 to L5.

In certain other embodiments, adeno-associated virus (AAV) is used to engineer the cells ex vivo or in vivo. AAV is a naturally occurring defective virus that requires a helper virus to produce infectious particles (
Muzyczka, Curr. Topics in Microbiol. Im
munol. , 158: 97 (1992)). AAV 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 can integrate,
Space for foreign DNA is limited to about 4.5 kb. Methods of generating and using such AAV are known in the art. For example, US Pat. No. 5,139
, 941, 5,173,414, 5,354,678, 5,
See 436, 146, 5,474,935, 5,478,745 and 5,589,377.

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

Another method of gene therapy is homologous recombination (see, eg, US Pat. No. 5,641,67).
No. 0, issued June 24, 1997; International Publication No. WO 96/29411, 199.
Published September 26, 2006; International Publication No. WO94 / 12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86
: 8932-8935 (1989); and Zijlstra et al., Nature.
342: 435-438 (1989)). Including. The method involves activation of a gene that is present in the target cell but is normally not expressed in that cell or is expressed at a lower level than desired.

Polynucleotide constructs are made using standard techniques known in the art. This construct contains a promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to the endogenous sequence to allow homologous recombination between the promoter-targeting sequence and the endogenous sequence. The targeting sequence is located sufficiently close to the 5'end of the desired endogenous polynucleotide sequence so that upon homologous recombination the promoter is operably linked to the endogenous sequence.

The promoter and targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains additional restriction enzyme sites at the 5'and 3'ends. Preferably, the 3'end of the first targeting sequence contains the same restriction enzyme sites as the 5'end of the amplified promoter, and the 5'end of the second targeting sequence contains the 3'end of the amplified promoter. Includes the same restriction sites as the termini. The amplified promoter and targeting sequences are digested and ligated together.

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

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

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

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

Any dosage form of any of the above polynucleotide constructs may be used, so long as the dosage form expresses one or more molecules in an amount sufficient to provide a therapeutic effect. . This includes direct needle injections, systemic injections, catheter injections, biolistic injectors, particle accelerators (ie "gene guns"), gel foam sponge depots, and other commercial depots.
pot) substances, osmotic pumps (eg Alza minipumps), solid (tablets or pills) pharmaceutical formulations for oral or suppository, and intra-operative 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 the portal vein resulted in gene expression of foreign genes in rat liver (Ka
neda et al., Science, 243: 375 (1989)).

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

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

Therapeutic compositions useful for systemic administration include a recombinant molecule of the present invention complexed with a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use in systemic administration include liposomes containing ligands that target the vehicle to a particular site.

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

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

Biological Activity The polynucleotides or polypeptides of the invention, or agonists or antagonists, 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 may be involved in diseases associated with their biological activity. Accordingly, the polynucleotides or polypeptides, or agonists or antagonists, can be used to treat associated diseases.

(Immune Activity) The polynucleotide, polypeptide, antibody, and / or agonist or antagonist of the present invention is activated, for example, by activating or inhibiting proliferation, differentiation, or recruitment (chemotactic) of immune cells. , May be useful in treating, preventing, and / or diagnosing diseases, disorders and / or conditions of the immune system. Immune cells develop through a process called hematopoiesis, producing myeloid cells (platelets, erythrocytes, neutrophils and macrophages) and lymphoid cells (B and T lymphocytes) from pluripotent stem cells. The etiology of these immune diseases, disorders and / or conditions can be genetic, somatic (eg cancer or some autoimmune disorders), acquired (eg due to chemotherapy or toxins) or infectious. possible. In addition, the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention can be used as markers or detectors of diseases or disorders of the particular immune system.

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

In addition, the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention may also be used to modulate hemostatic activity (stop bleeding) or thrombolytic activity (clot formation). . Polynucleotides or polypeptides of the invention and / or agonists or antagonists are used to increase the hemostatic or thrombolytic activity, for example, to treat diseases, disorders and / or conditions of blood coagulation (eg fibrinogen-free). Blood, platelet deficiency), blood platelet diseases, disorders and / or conditions (eg thrombocytopenia), or wounds resulting from trauma, surgery or other causes may be treated or prevented. Alternatively,
Polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the invention that can reduce hemostatic or thrombolytic activity can be used to inhibit or lyse blood clots. These molecules may be important in the treatment or prevention of heart attack (infarction), stroke or scarring.

The polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the invention may also be useful in treating, preventing and / or diagnosing autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign substances by immune cells. This improper recognition triggers an immune response that results in the destruction of host tissue. Therefore, an immune response, especially T cell proliferation,
Administration of the polynucleotides and polypeptides of the invention that can inhibit differentiation or chemotaxis can be an effective treatment in the prevention of autoimmune disorders.

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

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

Additional autoimmune disorders that can be treated, prevented, and / or diagnosed with the compositions of the present invention include, but are not limited to, the following: Chronic active hepatitis. (Eg, often characterized by smooth muscle antibodies), primary biliary cirrhosis (eg, often characterized by mitochondrial antibodies), other endocrine disorders (eg, in some cases often by specific tissue antibodies) , Vitiligo (often characterized by melanocyte antibodies), vasculitis (often characterized by Ig and complement in the vascular wall and / or low serum complement), post MI (eg , Often characterized by myocardial antibodies, open heart syndrome (eg, to myocardial antibodies) Thus often characterized), urticaria (eg, often characterized by IgG and IgM antibodies to IgE), atopic dermatitis (eg, often characterized by IgG and IgM antibodies to IgE), asthma ( For example, often characterized by IgG and IgM antibodies to IgE), and many other inflammatory, granulomatous, degenerative and atrophic disorders.

In a preferred embodiment, the autoimmune diseases and disorders and / or conditions associated with the diseases and disorders listed above are, for example, antagonists, or agonists, polypeptides or polynucleotides or antibodies of the invention. It is used to treat, prevent, and / or diagnose.

In certain preferred embodiments, the polynucleotides, polypeptides, and / or agonists or antagonists of the invention are for boosting immunoreactivity in B cell and / or T cell immunodeficient individuals. It can be used as a drug.

B-cell immunodeficiencies that can be ameliorated or treated by administering a polynucleotide or polypeptide of the invention, and / or agonists thereof include, but are not limited to: severe Combined immunodeficiency (SCI
D) -X-linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficient), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infant agammaglobulinemia Disease, acquired agammaglobulinemia, adult-onset agammaglobulinemia, late-onset agammaglobulinemia, abnormal gammaglobulinemia, hypogammaglobulinemia, transient infant hypogammaglobulinemia, nonspecific Hypogammaglobulinemia, agammaglobulinemia, unclassified immunodeficiency (CVI) (acquired), Viscott-Aldrich syndrome (WAS), X-linked immune deficiency with excess IgM, non-excessive IgM X-linked immune deficiency, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), normal Antibody deficiency with Ig or elevated Ig, immune deficiency with thymoma, Ig heavy chain deficiency, κ chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, regression agammaglobulinemia (Swiss type), reticulohypoplasia, neonatal neutropenia, severe congenital leukopenia, thymic lymphadenopathy with immune deficiency-hypoplasia or dysplasia, ataxia-telangiectasia. , Short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP),
Nezeroff syndrome complex immunodeficiency with Ig, purine nucleoside phosphorylase deficiency (PNP), MHC class II deficiency (deficient lymphocyte syndrome) and severe combined immunodeficiency.

T cell disorders that may be alleviated or treated by administering a polypeptide or polynucleotide of the invention, and / or an agonist thereof include, but are not limited to, eg, DiGeorge malformation, Thymic hypoplasia, 3rd and 4th pharyngeal sac syndrome, 22q11.2 deficiency, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4 + T lymphocyte depletion, immune deficiency with marked T cell deficiency ( Unspecified), and unspecified immune deficiency of cell-mediated immunity. In certain embodiments, the DiGeorge malformation or condition associated with the DiGeorge malformation is alleviated or treated, eg, by administering a polypeptide or polynucleotide of the invention, or an antagonist or agonist thereof.

Other immunodeficiency diseases that can be ameliorated or treated by administering the polypeptides or polynucleotides of the invention, and / or their agonists or antagonists, include severe combined immunodeficiency (SCID; eg, X-linked). S
CID, autosomal SCID, and adenosine deaminase deficiency), ataxia-telangiectasia, viscot-oldrich syndrome, short-limb (short-).
Liber) dwarfism, X-linked lymphocytosis syndrome (XLP), Nezerov's syndrome (eg, purine nucleoside phosphorylase deficiency), MHC class II deficiency, but is not limited thereto. In certain embodiments, ataxia-telangiectasia or conditions associated with ataxia-telangiectasia can be ameliorated or treated by administering a polypeptide or polynucleotide of the invention, and / or an agonist thereof. .

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

Similarly, allergic reactions and conditions such as asthma (especially allergic asthma)
Or other respiratory disorders) may also be treated with the polypeptides, antibodies, polynucleotides of the invention, and / or their agonists or antagonists,
It can be prevented and / or diagnosed. In addition, these molecules can be used to treat, prevent and / or diagnose anaphylaxis, hypersensitivity to antigenic molecules, or blood group mismatches.

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

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

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

The polypeptides, antibodies, polynucleotides and / or agonists or antagonists of the invention can be used to treat, detect, and / or prevent infectious agents. For example, by increasing the immune response, especially by increasing the proliferation, activation and / or differentiation of B and / or T cells,
Infectious diseases can be treated, detected, and / or prevented. The immune response can be augmented by either augmenting an existing immune response or eliciting a new immune response. Alternatively, the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the invention may also directly inhibit infectious agents (as described in the Infectious Agents Application Section, etc.) without the inevitable elicitation of an immune response. You can

Further preferred embodiments of the present invention include, but are not limited to, the use of polypeptides, antibodies, polynucleotides and / or agonists or antagonists in the following applications: boosting the immune system, one or more. Increase in the amount of antibodies (eg, IgG, IgA, IgM, and IgE) of high affinity antibody production (eg, IgG, Ig
A, IgM and IgE) and / or animals for increasing immune response (eg, mouse, rat, rabbit, hamster, guinea pig, pig, miniature pig, chicken, camel, goat, horse, cow, sheep, Administration to dogs, cats, non-human primates, and humans, most preferably humans).

A reconstituted or partially reconstituted immune system that is unable to produce a functional endogenous antibody molecule or has an otherwise immunocompromised endogenous immune system (E.g., PCT Publication No. WO98 / 24893, WO96) capable of producing human immunoglobulin molecules by the means of. / 34096
, WO 96/33735, and WO 91/10741).

[0399]   A vaccine adjuvant that enhances immune responsiveness to a specific antigen.

[0400]   Adjuvants for enhancing tumor-specific immune responses.

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

Adjuvants for increasing the anti-bacterial or anti-fungal immune response. Anti-bacterial or anti-fungal immune responses that may be augmented using the compositions of the present invention as an adjuvant include bacterial or fungal, and those described herein or otherwise known in the art. Included are bacteria or fungi associated with the disease or condition. In a particular embodiment, the composition of the invention comprises a bacterial or fungal, disease,
Alternatively, it is used as an adjuvant to increase the immune response to a condition, which is selected from the group consisting of tetanus, diphtheria, botulism, and meningitis B. In certain preferred embodiments, the composition of the invention comprises a bacterial or fungal, disease, or symptom, which is a Vibrio cholerae, Myco.
Bacterium leprae, Salmonella typhi, Sa
lmonella paraphyphi, Meisseria meningi
tidis, Streptococcus pneumoniae, Group
B streptococcus, Shigella spp. , Enter
otoxigenic Escherichia coli, Enterohe
morrhagic E. coli, Borrelia burgdorfer
i, and Plasmodium (selected from the group consisting of malaria)) as an adjuvant for increasing the immune response.

Adjuvants for increasing antiparasitic immune response. Anti-parasitic immune responses that may be augmented using the compositions of the present invention as an adjuvant are associated with parasites and diseases or conditions described herein or otherwise known in the art. Parasites are included. In a particular embodiment, the compositions of the invention are used as an adjuvant to increase the immune response against parasites. In another particular embodiment, the composition of the invention is used as an adjuvant for increasing the immune response to Plasmodium (malaria).

[0404]   As a stimulator of B cell responsiveness to pathogens.

[0405]   As an activator of T cells.

[0406]   As a drug that increases an individual's immune status before receiving immunosuppressive therapy.

[0407]   As a drug for inducing higher affinity antibodies.

[0408]   As a drug to increase serum immunoglobulin levels.

[0409]   As a drug to promote recovery of immunocompromised individuals.

[0410]   As a drug to boost immune responsiveness among older populations.

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

As a drug for boosting immune responsiveness among individuals with an acquired defect in B cell function. HIV acquired infections, AIDS, bone marrow transplants, and B cells include conditions that result in an acquired loss of B cell function that can be ameliorated or treated by administering a polypeptide, antibody, polynucleotide and / or agonist or antagonist thereof. Examples include, but are not limited to, chronic lymphocytic leukemia (CLL).

As an agent for boosting immune responsiveness between individuals with transient immunodeficiency. Viral infections (eg, influenza) include conditions that result in a transient immunodeficiency that can be ameliorated or treated by administering a polypeptide, antibody, polynucleotide and / or agonist or antagonist thereof.
Recovery from malnutrition, malnutrition-related conditions, recovery from infectious mononucleosis or stress-related conditions, recovery from measles, recovery from blood transfusions, recovery from surgery, but not limited to these. Not done.

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

[0415] The individual's immune system is responsive to the TH1 cellular response, as opposed to the humoral response (ie, TH
As a drug for directing the occurrence of 2).

As a means of inducing tumor growth and thus making the tumor more sensitive to anti-neoplastic agents. For example, multiple myeloma is a slow dividing cell division.
), And is therefore refractory to virtually all antitumor regimens. If these cells were grown more rapidly, their sensitivity profile would probably change.

AIDS, chronic lymphocytic disorders and / or unclassified immunodeficiency disorders (Comm
as a stimulator of B cell production in pathologies such as on Variable Immunology.

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

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

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

[0421]   As a means of activating T cells.

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

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

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

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

[0426]   All of the above applications may be applied to veterinary medicine.

The antagonists of the present invention include, for example, binding and / or inhibitory antibodies, antisense nucleic acids, ribozymes. These abolish many of the activities of the above ligands and are expected to find clinical or practical applications such as:

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

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

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

[0431]   As an inhibitor of graft-versus-host disease or graft rejection.

ALL, Hodgkin's disease, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, plasmacytoma, multiple myeloma, Burkitt's lymphoma and EBV convertible (EBV-tr
Treatment for B cell and / or T cell malignancies such as disorders.

Monoclonal hypergammaglobulinemia of undetermined significance (monoclona)
lgammopathy of unterminated signature
ance) (MGUS), Waldenström's disease, associated idiopathic monoclonal hypergammaglobulinemia, and plasmacytoma,
Treatment for chronic hypergammaglobulinemia events.

[0434]   Treatment to reduce cell proliferation of large B cell lymphoma.

[0435]   A means of reducing the involvement of B cells and Ig associated with chronic myelogenous leukemia.

[0436]   Immunosuppressants.

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

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

Agonists and antagonists can be used in compositions with a pharmaceutically acceptable carrier, for example, as described above.

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

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

The agonists and / or antagonists of the invention also have use in stimulating wound and tissue repair, stimulating angiogenesis, stimulating repair of vascular or lymphatic diseases or disorders. In addition, the agonists and antagonists of the invention can be used to stimulate regeneration of mucosal surfaces.

In a particular embodiment, the polynucleotide or polypeptide, and / or
Alternatively, the agonists are used to treat or prevent disorders characterized by primary or acquired immunodeficiency, defective serum immunoglobulin production, recurrent infections and / or immune system dysfunction. In addition, the polynucleotides or polypeptides, and / or their agonists may be used for infection of joints, bones, skin and / or parotid glands, blood-borne infections (eg sepsis, meningitis, septic arthritis and / or bone marrow). Inflammation), autoimmune diseases (eg, autoimmune diseases as disclosed herein), inflammatory disorders, and malignant diseases, and / or any of these infections, diseases and / or malignant diseases. Disease or disorder or condition (CVID, other primary immunodeficiency, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (eg, severe zoster) Herpes) and / or pneumocystis), but is not limited thereto.

In another embodiment, the polynucleotides, polypeptides, antibodies, and / or agonists or antagonists of the invention are non-classified immunodeficiency (C
Ommon Variable Immunology ("CV
ID "; also known as" acquired agammaglobulinemia "and" acquired hypogammaglobulinemia ") or an individual with a subset of this disease is used to treat and / or diagnose.

[0445] In certain embodiments, the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the invention induce (1) cancer or neoplasms, and (2) autoimmune cells or tissue-associated cancers or neoplasms. Treatment, diagnosis, and / or
Or it can be used to prevent. In a preferred embodiment, the polynucleotides, polypeptides, antibodies and / or agonists or antagonists of the invention conjugated to a toxin or radioisotope, as described herein, induce acute myelogenous leukemia. It can be used to treat, diagnose, and / or prevent. In a further preferred embodiment, the polynucleotide, polypeptide, antibody, and / or agonist or antagonist of the invention conjugated to a toxin or radioactive isotope, as described herein, is used in chronic bone marrow. Treatment of leukemia, multiple myeloma, non-Hodgkin's lymphoma, and / or Hodgkin's disease,
It can be used to diagnose, diagnose, and / or prevent.

In another particular embodiment, the polynucleotides or polypeptides of the invention and / or agonists or antagonists are used to produce selective Ig
A deficiency, myeloperoxidase deficiency, C2 deficiency, ataxia-telangiectasia, Di
George malformation, unclassifiable immunodeficiency (CVI), X-linked agammaglobulinemia, severe combined immunodeficiency (SCID), chronic granulomatosis (CGD), and Wisk
The Ott-Aldrich syndrome may be treated, diagnosed, predicted, and / or prevented.

Examples of autoimmune disorders that may be treated or detected include Addison's disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's syndrome, Graves. Disease, multiple sclerosis, myasthenia gravis, neuritis, ocular pain, bullous pemphigoid, pemphigus, multiple endocrine disorders, purpura, Reiter's disease, Stiffman syndrome, autoimmune thyroiditis, Systemic lupus erythematosus, autoimmune lung inflammation, Guyan-Barre syndrome, insulin-dependent diabetes mellitus,
And autoimmune inflammatory eye diseases, but are not limited thereto.

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

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

Furthermore, the polynucleotides, polypeptides and / or antagonists of the invention may affect apoptosis, thus the polynucleotides, polypeptides and / or antagonists of the invention may increase cell survival or inhibit apoptosis. It is useful in treating many related diseases. For example, diseases associated with increased cell survival or inhibition of apoptosis that may be treated or detected by the polynucleotides, polypeptides and / or antagonists of the invention include: cancer (eg, follicular lymphoma, p53). Cancers with mutations and hormone-dependent tumors (colorectal cancer, heart tumor, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, gastric cancer, neuroblastoma, mucus Tumor, myoma, lymphoma, endothelium, osteoblastoma, giant cell tumor of bone, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer)); Immune disorders (eg, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, clones) , Polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (eg, herpesvirus, poxvirus and adenovirus), inflammation, graft-versus-host disease, acute graft rejection, and Chronic graft rejection. In a preferred embodiment, the polynucleotide of the present invention,
The polypeptides and / or antagonists are used to inhibit the growth, progression, and / or metastasis of cancers, especially those listed above.

Additional diseases or conditions associated with cell survival that may be treated or detected by the polynucleotides, polypeptides and / or antagonists of the invention include proliferation and / or metastasis of malignant tumors and associated disorders such as: But not limited to: leukemia (acute leukemia (eg, acute lymphocytic leukemia, acute myelogenous leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and Erythroleukemia)), and chronic leukemia (eg, including chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma (eg, Hodgkin's disease and non-Hodgkin's disease), Multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, and solid tumors (sarcoma and And carcinomas (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, periosteoma, mesothelioma, Ewing tumor, Leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer,
Ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial progenitor carcinoma, renal cell carcinoma, hepatocyte Cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung cancer,
Small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendrocyte Gliomas, menangiomas, melanomas, neuroblastomas, and retinoblastomas.

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

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

Similarly, other hyperproliferative disorders can also be treated or detected by the polynucleotides, polypeptides, and / or antagonists of the invention. Examples of such hyperproliferative disorders include, but are not limited to, hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemia, purpura, sarcoidosis, Sézary syndrome, Waldenström. Macroglobulinemia,
Gaucher disease, histiocytosis, and any other hyperproliferative disease, as well as neoplasms found in the organ systems listed above.

Hyperproliferative Disorders Polynucleotides or polypeptides of the invention, or agonists or antagonists, can be used to treat, prevent and / or diagnose hyperproliferative diseases, disorders, including neoplasms. The polynucleotides or polypeptides of the invention, or agonists or antagonists, may inhibit the growth of this disorder via direct or indirect interactions. Alternatively, the polynucleotides or polypeptides of the invention, or agonists or antagonists, can proliferate other cells that can inhibit hyperproliferative disorders.

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

Examples of hyperproliferative diseases, disorders and / or conditions that can be treated, prevented and / or diagnosed by the polynucleotides or polypeptides of the invention, or agonists or antagonists, include colon, abdomen, bone, breast, Digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal gland, parathyroid gland, pituitary gland, testis, ovary, thymus, thyroid), eyes, head and neck, nerves (central and peripheral), lymphatic system, pelvis, skin, Includes, but is not limited to, neoplasms located in soft tissue, spleen, rib cage, and genitourinary organs.

Similarly, other hyperproliferative diseases, disorders and / or conditions may also be treated, prevented and / or diagnosed with a polynucleotide or polypeptide of the invention, or an agonist or antagonist. Examples of such hyperproliferative diseases, disorders and / or conditions include hypergammaglobulinemia, lymphoproliferative disorders, disorders and / or conditions, paraproteinemia, purpura, sarcoidosis,
Sézary syndrome, Waldenström macroglobulinemia, Gaucher's disease, histiocytosis, as well as any other hyperproliferative disorders, as well as neoplasms located in the organ systems listed above, It is not limited to them.

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

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

Another embodiment of the invention provides a method of treating or preventing a cell proliferative disease, disorder and / or condition in an individual, the method comprising treating cells that are abnormally proliferating. The step of administering one or more active gene copies is included. In a preferred embodiment, the polynucleotide of the present invention is a DNA construct containing a recombinant expression vector effective in expressing the DNA sequence encoding the above polynucleotide. In another preferred embodiment of the invention, a DNA construct encoding a polynucleotide of the invention is inserted into a cell and treated with a retrovirus (or, more preferably, an adenovirus vector). G J. Nabel et al., PNAS 1999 96: 324, incorporated herein by reference.
-326). In the most preferred embodiment, the viral vector is deficient and does not transform non-proliferating cells, only proliferating cells. Further, in a preferred embodiment, the polynucleotide of the invention inserted into proliferating cells, alone or in combination with or in fusion with other polynucleotides, is then subjected to external stimulation (i.e., Magnetic properties, certain small molecules, chemicals, or drug administration, etc.). This stimulus acts on a promoter upstream of the above polynucleotide to induce expression of the encoded protein product. In this way, the beneficial therapeutic effects of the present invention can be clearly modulated (ie, to increase, decrease, or inhibit expression of the polynucleotides of the present invention) based on the external stimuli described above.

The polynucleotides of the present invention may be useful in suppressing the expression of oncogenic genes or antigens. By "suppressing the expression of oncogenic genes", suppression of gene transcription, degradation of gene transcript (pre-message RNA), inhibition of splicing, messenger RNA destruction, and prevention of post-translational modification of proteins , Intended to destroy a protein, or inhibit the normal function of a protein.

For topical administration to aberrantly proliferating cells, the polynucleotides of the invention may be administered by any method known to those of skill in the art, including transfection, electroporation, cellular Microinjection, including, but not limited to, lipofectin, or naked polynucleotides in vehicles such as liposomes, or any other method described throughout this specification. The polynucleotides of the present invention can be used in known gene delivery systems (eg, retroviral vectors (Gilboa,
J. Virology 44: 845 (1982); Hocke, Nature.
320: 275 (1986); Wilson et al., Proc. Natl. Aca
d. Sci. USA. 85: 3014); vaccinia virus system (Chakra)
barty et al., Mol. Cell Biol. 5: 3403 (1985)) or other efficient DNA delivery systems (Yates et al., Nature 313: 812 (
1985))). These references are exemplary only and incorporated herein by reference. Retroviruses or adenoviruses known to those of skill in the art to specifically deliver to, or transfect, and to leave non-dividing cells in cells that are abnormally proliferating (eg, in the art or It is preferred to utilize a delivery system (as described herein). 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 invention, such retroviral delivery systems are utilized to target the above genes and constructs to aberrantly proliferating cells and leave non-dividing normal cells.

The polynucleotides of the present invention can be used to direct cell proliferative disorders in internal organs, body cavities, etc. by the use of imaging devices that are used to direct the injection needle directly to the site of disease.
It can be delivered directly to the disease site. The polynucleotides of the present invention may also be administered to the disease site during surgical intervention.

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

Any amount of a polynucleotide of the invention can be administered so long as the amount has a biologically inhibitory effect on the growth of treated cells. Moreover, more than one polynucleotide of the invention can be administered at the same site at the same time. "
By “biologically inhibitory” is meant partial or total growth inhibition as well as a decrease in the rate of proliferation or growth of cells. A biologically inhibitory dose may be used to assess the effect of a polynucleotide of the invention on the growth of abnormally proliferating cells in the target malignant or tissue culture, tumor growth in animals and cell culture, or It can be determined by any other method known to those skilled in the art.

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

An overview of the ways in which the antibodies of the invention can be used therapeutically is to provide a polynucleotide or polypeptide of the invention, either locally or systemically in the body, or directly to the cytotoxicity of the antibody (eg, Binding via complement mediated (CDC) or effector cell mediated (ADCC). Some of these approaches are described in more detail below. Given the teachings provided herein, one of skill in the art would know how to use the antibodies of the invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the invention have or develop a disease, disorder and / or condition of cell proliferation and / or differentiation as described herein. It is useful for treating, preventing and / or diagnosing an existing subject. Such treatment involves administering a single dose or multiple doses of the antibody, or a fragment, derivative, or conjugate thereof.

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

[0471]   A polypeptide or polynucleotide of the invention, a fragment thereof or
A high affinity and / or potent in vivo inhibitory antibody against the region and / or
The use of a neutralizing antibody is useful for
Immunity to diseases, disorders and / or conditions (including fragments thereof)
Preferred for both assay and its treatment. Such an antibody, Fragme
The component, or region, is preferably a polynucleotide or polypeptide (that
(Including fragments thereof). Preferred binding affinity is 5x
10^-6M, 10^-6M, 5 x 10^-7M, 10^-7M, 5 x 10^-8M, 10 ^-8 M, 5 x 10^-9M, 10^-9M, 5 x 10^-10M, 10^-10M, 5x
10^-11M, 10^-11M, 5 x 10^-12M, 10^-12M, 5 x 10^{-1 Three} M, 10^-13M, 5 x 10^-14M, 10^-14M, 5 x 10^-15M, oh
And 10^-15Includes affinities with a dissociation constant or Kd of less than M.

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

The polypeptides of the invention (including protein fusions), or fragments thereof, may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. The polypeptides described above may be used, for example, to induce apoptosis of proliferative cells and tissues either directly or indirectly, eg, 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 (
Schulze-Osthoff K, et al., Incorporated herein by reference,
Eur J Biochem 254 (3): 439-59 (1998))). Further, in another preferred embodiment of the present invention, the above-mentioned polypeptide is mediated by other mechanisms (for example, in the activation of other proteins that activate apoptosis) or by expressing the protein alone or as a small molecule drug. Alternatively, apoptosis can be induced either by stimulation with either an adjuvant (eg, apoptonin, galectin, thioredoxin, anti-inflammatory protein) (eg, Mutat Res, incorporated herein by reference). 400 (1-2): 447-55 (1
998), Med Hypotheses. 50 (5): 423-33 (199
8), Chem Biol Interact. Apr 24; 111-112.
23-34 (1998)), J Mol Med. 76 (6): 402-12.
(1998), Int J Tissue React; 20 (1): 3-15.
(1998)).

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

In another embodiment, the invention comprises a composition comprising a polypeptide of the invention (eg, a composition comprising a polypeptide antibody, a heterologous nucleic acid, a toxin, or a prodrug related to the polypeptide or a heterologous polypeptide). There is provided a method of delivering an article to a targeted cell expressing a polypeptide of the invention. Polypeptides or polypeptide antibodies of the invention can be attached to heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs through hydrophobic, hydrophilic, ionic and / or covalent interactions.

Polypeptides of the invention, protein fusions thereto, or fragments thereof, may be used directly against proliferative antigens and immunogens (eg, when the polypeptide of the invention is "vaccinated") as described above. An immune response to respond to an antigen and an immunogen of E. coli or indirectly (eg, in activating the expression of proteins known to enhance the immune response (eg, chemokines)). And are useful in enhancing the immunogenicity and / or antigenicity of proliferating cells or tissues.

Cardiovascular Disorders Polynucleotides or polypeptides, or agonists or antagonists of the invention are used to treat cardiovascular diseases, disorders and / or conditions, including peripheral arterial disease such as limb ischemia. , May be prevented and / or diagnosed.

Cardiovascular diseases, disorders and / or conditions include arterial fistula
cardiovascular abnormalities such as o-arterial fistula, arteriovenous fistula, cerebral arteriovenous birth defects, congenital heart defects, pulmonary valve atresia, and Scimitar syndrome. Congenital heart defects include aortic coarctation, tricentric heart and coronary vascular malformations.
el anomalies), cross heart, right thoracic heart, patent ductus arteriosus (patent)
dactus arteriosus), Ebstein's malformation, Eisenmenger complex, left ventricular underdevelopment syndrome, left thoracic heart, tetralogy of Fallot, transposition of the great vessels, right ventricle bilateral large veins, tricuspid regurgitation, arterial duct remnants Remnants, and heart septal defects (heart)
septal defects (eg, aortopulmonary pulmonary septal defect (aor
topulmonal septal defect, endocardial bed defect, Luthambache syndrome, trilogy of Fallot, ventricular septal defect (ventricu)
lar heart septeral defects)).

Cardiovascular diseases, disorders and / or conditions also include arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, intracardiac. Membranitis (including bacterial), cardiac aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, hydrocephalus, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, infarction Posterior heart rupture,
Ventricular septal rupture, heart valve disease, myocardial disease, myocardial ischemia, pericardial effusion, pericarditis (including infarct and tuberculosis), pericardial disease, postpericardiotomy syndrome, right heart disease, Rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications (cardiovascu)
lar pregnancy complications, scimitar syndrome, cardiovascular syphilis, and cardiovascular tuberculosis
heart diseases such as culosis).

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, premature contraction, Adams-Stokes syndrome, leg block, sinoatrial block, long QT syndrome (long).
QT syndrome), collateral contraction, Lone-Ganning-Levine syndrome, Maheme-type pre-excita
tion syndrome), Wolff-Parkinson-White syndrome, sinus dysfunction syndrome, tachycardia, and ventricular fibrillation. As tachycardia, paroxysmal tachycardia,
Supraventricular tachycardia, ventricular intrinsic rhythm promotion, atrioventricular nodal reentry tachycardia (atrioventri
circular nodal reentry tachycardia), ectopic atrial tachycardia, ectopic junction tachycardia, sinoatrial nodal reentry tachycardia
dal reentry tachycardia), sinus tachycardia, torsade
De pointe, and ventricular tachycardia.

Heart valve diseases include aortic insufficiency, aortic stenosis, and murmur (hea).
r murmurs), aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve dysfunction, mitral valve stenosis, pulmonary valve atresia, pulmonary valve dysfunction, pulmonary valve stenosis, tricuspid Includes atresia, tricuspid dysfunction, and tricuspid stenosis.

Cardiomyopathy includes alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, subvalvular aortic stenosis, subvalvular pulmonary stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, intracardiac Membrane fibroelasticity, endocardial myocardial fibrosis, Keens syndrome, myocardial reperfusion injury, and myocarditis.

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

Cardiovascular diseases also include aneurysms, dysplasias, hemangiomatosis, bacterial hemangiomas, Hippel-Lindau Disease.
), Klipel-Tornone-Weber syndrome, Sturgi-Weber syndrome,
Vasomotor edema, aortic disease, Takayasu arteritis, aortitis, Leuriche syndrome,
Arterial occlusion disease, arteritis, endarteritis, polyarteritis nodosa, cerebrovascular disease, disorder and / or condition, diabetic vascular disorder, diabetic retinopathy, embolism, thrombosis, tip Erythema, hemorrhoids, hepatic vein occlusion disorder, hypertension, hypotension, ischemia, peripheral vascular disorder, phlebitis, pulmonary vein occlusion disease, hypertension, hypotension, ischemia, peripheral vascular disease, phlebitis, pulmonary vein occlusion disease , Raynaud's disease, CREST syndrome, retinal vein occlusion, scimitar syndrome, superior vena cava syndrome, telangiectasia, ataxia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, dilated meandering Vascular diseases such as veins, varicose ulcers, vasculitis, and venous insufficiency are included.

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

Arterial occlusive disease includes arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasia, mesenteric vascular occlusion, Moyamoya disease, renal artery occlusion, retinal artery occlusion, and occlusive thrombosis. Sexual vasculitis.

Cerebrovascular diseases, disorders and / or conditions include carotid artery disease, cerebral amyloid vasculopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous birth defects, cerebral artery disease, cerebral artery Embolism and thrombosis, carotid thrombosis, sinus thrombosis, Wallenberg syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage (subara)
xhnoid hemorrhage), cerebral infarction, cerebral ischemia (including transient)
, Subclavian stealing syndrome, periventricular leukomalacia
eukomalacia), vascular headaches, cluster headaches, migraine headaches, and vertebrobasilar dysfunction.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, toe cyanosis syndrome, fat embolisms, pulmonary embolisms, and thromboembolisms. As thrombosis, coronary artery thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis,
Includes sinus thrombosis, Wallenberg syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, and partition syndrome (compartme).
nt syndrome, anterior compar
ment syndrome), myocardial ischemia, reperfusion injury, and peripheral limb ischemia. Vasculitis includes aortitis, arteritis, Behcet.
t) Syndrome, Churg-Strauss Syndrome, Mucocutaneous Lymph Node Syndrome, Obstructive Thrombotic Vasculitis, Hypersensitivity Vasculitis, Schoenlein-Henoho Purpura (Schoenl)
ein-Henoch purpura), allergic cutaneous vasculitis and Wegener's granulomatosis.

The polynucleotides or polypeptides of the invention, or agonists or antagonists, are particularly effective for the treatment of 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 injection, biolistic injection, particle accelerators, gel foam sponge depots, other commercial depot materials, osmotic pumps, solid pharmaceutical formulations for oral or suppository, intraoperative decanting or topical application, aerosol delivery. But is not limited to these. Such methods are known in the art.
The polypeptides of the present invention are described in more detail below in therapeutic agents (Theraps).
eutic). Methods of delivering the polynucleotides of the invention are described in more detail herein.

Anti-Angiogenic Activity The naturally occurring equilibrium between an endogenous stimulator and an inhibitor of angiogenesis is the equilibrium that governs the inhibitory effects. Rastinejad et al., Cell 56
: 345-355 (1989). In rare cases where neovascularization occurs under normal physiological conditions (eg, wound healing, organ regeneration, embryogenesis, and female reproductive processes), angiogenesis is tightly regulated and spatial and temporal. It is specified. Under pathological angiogenic conditions (eg, which characterize solid tumor growth), there is no control over these controls. Unregulated angiogenesis becomes pathological and maintains the progression of many neoplastic and non-neoplastic diseases. Many serious diseases are dominated by abnormal neovascularization, including the growth and metastasis of solid tumors, arthritis, some types of ocular disorders and psoriasis. For example, Mo
ses et al., Biotech. 9: 630-634 (1991); Folkman.
N. et al. Engl. J. Med. 333: 1757-1763 (1995);
Auerbach et al. Microvasc. Res. 29: 401-411
(1985); Folkman, Advances in Cancer Re.
search, Klein and Weinhouse, Academic P
less, New York, pp. 175-203 (1985); Patz, Am.
． J. Opthalmol. 94: 715-743 (1982); and Fol.
See review by Kman et al., Science 221: 719-725 (1983). In many pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data has been accumulated that suggests that solid tumor growth depends on angiogenesis. Folkman and Klagsbrun, S
science 235: 442-447 (1987).

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

In yet another aspect, the polynucleotides, polypeptides, antagonists and / or agonists can be utilized to treat, prevent and / or diagnose surface morphologies of bladder cancer, eg, by intravesical administration. Polynucleotides, polypeptides, antagonists and / or agonists can be delivered directly to the tumor or near the tumor site via injection or catheter. Of course, as the skilled artisan will appreciate, the appropriate mode of administration will vary with the cancer to be treated. Other modes of delivery are discussed herein.

Polynucleotides, polypeptides, antagonists and / or agonists are useful in treating, preventing and / or diagnosing other diseases, disorders and / or conditions (including angiogenesis) in addition to cancer. Can be. These disorders, diseases and / or conditions include, but are not limited to:
Benign tumors (eg, hemangiomas, acoustic neuromas, neurofibromas, trachoma, and pyogenic granulomas); arteriosclerotic plaques; angiogenic diseases of the eye (eg, diabetic retinopathy, immature retinopathy, macular degeneration, corneas) Transplant rejection, neovascular glaucoma, posterior lens fibrosis, rubeosis, retinoblastoma, uvietis and pterygium of the eye (Pter).
ygia) (abnormal blood vessel growth)); rheumatoid arthritis; psoriasis; delayed wound healing;
Endometriosis; Angiogenesis; Granulation; Hyperplastic scar (keloid); Pseudoarthritis fracture; Scleroderma; Trachoma; Vascular adhesion; Myocardial angiogenesis; Coronary coronary
cerebral collaterals; arteriovenous malformations; ischemic extremity angiogenesis; Osler-Webber syndrome; plaque neovascularization; telangiectasia; hemophilic joints; angiofibromas; Fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.

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

In one embodiment of the invention, polynucleotides, polypeptides, antagonists and / or agonists are directly injected into hyperplastic scars or keloids to prevent the progression of these lesions. This treatment is of particular value in the prophylactic treatment of conditions known to result in the development of hyperplastic scars and keloids (eg burns), and preferably at the time the proliferative phase progresses (of the initial injury). About 14 days later) but before the onset of hyperplastic scars or keloids. As noted above, the present invention also treats, prevents and / or treats neovascularization disorders of the eye, including corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, post lens fibroplasia and macular degeneration. Or provide a method for diagnosing.

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

Accordingly, in one aspect of the invention, corneal neovascularization (comprising the step of administering to the patient a therapeutically effective amount of a compound (described above) to the cornea such that the formation of blood vessels is inhibited. Methods are provided for treating neovascularization disorders of the eye, including corneal transplant neovascularization). Briefly, the cornea is a tissue that normally lacks blood vessels. However, in certain pathological conditions, capillaries may extend from the limbal pericorneal plexus to the cornea. When the cornea is vascularized, it also becomes clouded, resulting in diminished vision of the patient. Complete loss of vision can occur when the cornea is completely opacitated. A wide variety of diseases, disorders and / or conditions can result in corneal neovascularization including, for example: corneal infections (eg trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes. (Eg, graft rejection and Stevens-Johnson syndrome), alkaline burns, trauma, inflammation (from any cause), toxic and nutritional deficiencies, and as a complication of wearing contact lenses.

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

In another embodiment, the above compounds may be injected directly into the corneal stroma by an ophthalmologist under guidance under a microscope. Although the preferred site of injection 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, dispersed between blood vessels and normal corneas). It is). In most cases, this is the limb (pe) to "protect" the cornea from advancing vessels.
rilimbic) corneal injection. This method can also be utilized immediately after corneal injury to prevent corneal neovascularization prophylactically. In this situation, the substance may be injected between the corneal lesion and its undesired potential blood supply limbus and injected into the perilimbal cornea. Such methods can also be utilized in a similar fashion to prevent capillary invasion of the transplanted cornea. In the sustained release form, infusion may only be required 2-3 times a year. Steroids may also be added to the infusion solution to reduce inflammation resulting from the injection itself.

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

In a particularly preferred embodiment of the invention, proliferative diabetic retinopathy affects the aqueous humor or the vitreous to increase local concentrations of polynucleotides, polypeptides, antagonists and / or agonists in the retina. It can be treated by injection. Preferably, this treatment should be initiated prior to the acquisition of the severe disease requiring photocoagulation.

In another aspect of the invention, a lens comprising administering to a 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 post-fibroproliferative disorders. The compounds may be administered locally via intravitreal injection and / or via intraocular implantation.

Further, disorders that can be treated, prevented and / or diseased with the polynucleotides, polypeptides, agonists and / or agonists include, but are not limited to: hemangiomas, arthritis, psoriasis, blood vessels. Fibromas, atherosclerotic plaques, delayed wound healing, granulation, hemophilic joints, hyperplastic scars,
Nonunion fractures, Osler-Weber syndrome, pyogenic granulomas, scleroderma, trachoma, and vascular adhesion.

In addition, disorders and / or conditions that may be treated with the polynucleotides, polypeptides, agonists and / or agonists include, but are not limited to: solid tumors, blood borne. Tumors (eg leukemia), tumor metastases, Kaposi's sarcoma, benign tumors (eg hemangiomas, acoustic neuromas, neurofibromas, trachoma and purulent granulomas), rheumatoid arthritis,
Psoriasis, ocular angiogenic disorders (eg diabetic retinopathy, retinopathy of prematurity, macular degeneration,
Corneal transplant rejection, neovascular glaucoma, post lens fibroplasia, rubeosis, retinoblastoma, and uvietis), delayed wound healing, endometriosis,
Angiogenesis, granulation, hyperplastic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesion, myocardial angiogenesis, coronary colla
, cerebral collaterals, arteriovenous malformations, ischemic extremity angiogenesis, Ausler-Weber syndrome, plaque neovascularization, telangiectasia, hemophilic joints,
Hemangiofibromas, fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control agents (by controlling menstruation, by preventing angiogenesis necessary for embryo implantation), pathogenesis Sexual consequences (eg scratch (Rochele mi
nia quintosa), ulcer (Helicobacter pylo)
ri), Bartonellosis and symptoms of bacterial hemangiomas).

In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered prior to or after sexual intercourse and fertilization occur, thus providing an effective method of birth control, perhaps Provide a "morning after" method. Polynucleotides, polypeptides, agonists and / or agonists can also be used in controlling menstruation or can be administered either as peritoneal lavage fluid 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 is
It can be utilized in a wide variety of surgical procedures. For example, in one aspect of the invention, the composition (eg, in the form of a spray or film) is used to separate normal surrounding tissue from malignant tissue and / or prevent the spread of disease to surrounding tissue. In order to do so, it can be utilized to coat or spray the area prior to tumor removal. In another aspect of the invention, the composition (eg, in the form of a spray) is delivered via an endoscopic procedure to coat a tumor or to inhibit angiogenesis at a desired location. obtain. In yet another aspect of the invention, a surgical mesh coated with an anti-angiogenic composition of the invention can be utilized in any procedure in which a surgical mesh can be utilized. For example, in one embodiment of the invention, a surgical mesh laden with an anti-angiogenic composition.
en) may be utilized during abdominal cancer resection surgery (eg, after colectomy) to provide support for the structure and to release certain amounts of anti-angiogenic factors.

In a further aspect of the invention, methods for treating a tumor resection site are provided. This method involves administering a polynucleotide, polypeptide, antagonist, and / or agonist to the margin of the tumor after resection to inhibit local recurrence of cancer and neovascularization at the site. Includes. 1 of the present invention
In one embodiment, the anti-angiogenic compound is administered directly to the tumor resection site (eg, the anti-angiogenic compound is used to swab, brush, or cut the resection margin of the tumor). Applied by coating in other ways).
Alternatively, the anti-angiogenic compound can be incorporated into known surgical pastes prior to administration. In a particularly preferred embodiment of the invention, the anti-angiogenic compound is applied after hepatectomy for malignant disease and after neurosurgery.

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

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

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

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

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

A wide variety of other anti-angiogenic factors may also be utilized in the context of the present invention. Representative examples include: Platelet Factor 4; Protamine Sulfate; Sulfated Chitin Derivatives (prepared from the queen crab shell) (Mur
ata et al., Cancer Res. 51: 22-26, 1991); sulfated polysaccharide peptidoglycan complex (SP-PG) (the function of this compound can be enhanced by the presence of steroids such as estrogen and tamoxifen citrate); staurosporine; Regulators of substrate metabolism (eg, proline analogs,
Cishydroxyproline, d, L-3,4-dehydroproline, thiaproline,
α, α-dipyridyl, including aminopropionitrile fumarate); 4-propyl-5- (4-pyridinyl) -2 (3H) -oxazolone; methotrexate;
Mitzantron; Heparin; Interferon; 2 Macroglobulin-serum; C
hIMP-3 (Pavloff et al., J. Bio. Chem. 267: 17321).
~ 17326, 1992); chymostatin (Tomkinson et al., Bioch.
em J. 286: 475-480, 1992); cyclodextrin tetradeca sulfate; eponomycin; camptothecin; fumagillin (Ingber
Et al., Nature 348: 555-557, 1990); sodium gold thiomalate ("GST"; Matsubara and Ziff, J. Clin. In.
vest. 79: 1440-1446, 1987); anti-collagenase-serum; α2-antiplasmin (Holmes et al., J. Biol. Chem. 262 (4).
): 1659-1664, 1987); Bisantoren (National Ca)
ncer Institute); Lobenzarit disodium (N- (2)-
Carboxyphenyl-4-chloroanthronic acid
ilic acid) disodium, or "CCA"; Takeuchi et al.,
Agents Actions 36: 312-316, 1992); thalidomide; Angostatic steroids; AGM-1470; carboxynaminolimidazole; and metalloproteinase inhibitors (eg, BB94).

Diseases at the Cellular Level For diseases associated with increased cell survival or inhibition of apoptosis that can be treated, prevented and / or diagnosed by the polynucleotides or polypeptides of the invention and / or antagonists or agonists. Include: cancer (eg, follicular lymphoma, carcinoma with p53 mutations, and hormone dependent tumors, including but not limited to: colon cancer, heart tumor, pancreatic cancer,
Melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, gastric cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelium, osteoblastoma, osteoclastoma, Osteosarcoma,
Chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune diseases, disorders and / or conditions (eg multiple sclerosis, Sjogren's syndrome, Hashimoto thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease) , Polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis), and viral infections (eg, herpesvirus, poxvirus and adenovirus), inflammation, graft-versus-host disease, acute graft rejection, And chronic graft rejection. In a preferred embodiment, the polynucleotides or polypeptides and / or agonists or antagonists of the invention are used to inhibit cancer growth, progression, and / or metastasis, especially in those listed above. It

Additional diseases or conditions associated with increased cell survival that can be treated, prevented and / or diagnosed with the polynucleotides or polypeptides of the invention, or agonists or antagonists, include malignant disease progression and / or metastasis and Related disorders include, but are not limited to, leukemia (acute leukemia (eg, acute lymphocytic leukemia, acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic ) And chronic leukemia (eg, chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma (eg, Hodgkin's disease and non-Hodgkin's disease). ), Multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, and solid Tumors, including but not limited to: sarcomas and carcinomas (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
Endotheliosarcoma, lymphangiosarcoma, lymphangioendothelioma, periosteal tumor, 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 adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchiogenic cancer, kidney Cell carcinoma,
Hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, Medulloblastoma, craniopharyngioma, ependymoma, pineocytoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and Retinoblastoma))).

Diseases associated with increased apoptosis that may be treated, prevented and / or diagnosed by the polynucleotides or polypeptides of the invention and / or agonists or antagonists include: AIDS; neurodegenerative diseases, Disorders and / or conditions (eg Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumors or prior related disorders); autoimmune diseases, disorders and / or conditions (eg , Multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis, and myelodysplastic syndromes (eg regeneration) Poor anemia), graft-versus-host disease, ischemic Injury (such as that caused by myocardial infarction, stroke and reperfusion injury),
Liver injury (eg, hepatitis-related liver injury, ischemia / reperfusion injury, cholestasis (cho)
lestosis (bile duct injury) and liver cancer); toxic-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.

Wound Healing and Epithelial Cell Proliferation In accordance with yet a further aspect of the invention, the polynucleotides or polypeptides of the invention and / or agonists or antagonists are used for therapeutic purposes.
For example, processes are provided for utilization to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and skin wound healing. A polynucleotide or polypeptide of the invention,
And agonists or antagonists may be clinically useful in stimulating wound healing, including: surgical wounds, excisional wounds, deep wounds (including dermal and epidermal damage), ocular tissue wounds, Dental tissue wounds, oral wounds, diabetic ulcers, skin ulcers, elbow ulcers, arterial ulcers, venous stasis ulcers, burns resulting from heat exposure or chemicals, and other abnormal wound healing Conditions (eg uremia, malnutrition, vitamin deficiency, and / or complications associated with systemic treatment with steroids, radiotherapy and anti-neoplastic drugs and antimetabolites. Polynucleotides or polypeptides of the invention, And agonists or antagonists can be used to promote skin recovery after skin loss.

The polynucleotides or polypeptides and / or agonists or antagonists of the invention are for increasing the adherence of skin grafts to the wound bed and for stimulating re-epithelialization from the wound bed. Can be used. The following are types of implants 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, autograft, autoepdermographic graft, avascular (avacu)
lar) graft, Blair-Brown graft, bone graft, embryonic tissue graft, dermal graft, delayed graft, skin graft, epidermal graft, fascia graft, full-thickness skin graft, xenograft. (Heterologous graft), xenograft (xenog)
Raft), homograft graft, proliferative graft, lamellar graft, reticular graft, mucosal graft, Olie-Tielsch graft, omental graft, patch implantation. Grafts, stalk-like grafts, penetrating grafts, split-thickness grafts (splits)
kin graft), thick split graft.
The polynucleotides or polypeptides and / or agonists or antagonists of the present invention can be used to promote skin strength and improve the appearance of aged skin.

The polynucleotides or polypeptides and / or agonists or antagonists of the present invention may also induce hepatocyte proliferation and lung, breast, pancreas, stomach, small intestine (
small intesting) and changes in epithelial cell proliferation in the large intestine. A polynucleotide or polypeptide of the invention,
And / or the agonist or antagonist is an epithelial cell (eg, sebocyte, hair follicle, hepatocyte, alveolar epithelial cell type II).
II pneumocyte), mucin-producing goblet cells, and other epithelial cells, and their ancestors contained in the skin, lungs, liver, and gastrointestinal tract). Polynucleotides or polypeptides of the invention, agonists and / or
Alternatively, the antagonist may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

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

The polynucleotides or polypeptides and / or agonists or antagonists of the present invention provide sufficient regeneration of skin (ie, hair follicles, sweat glands, and sebaceous glands) in full and partial thickness skin defects, including burns. Regrowth), and may be further used in the treatment of other skin defects such as psoriasis. The polynucleotides or polypeptides of the present invention, and agonists and / or antagonists, affect the epidermolysis bullosa, an endogenous dermis that causes frequent open and painful blisters by promoting re-epithelialization of these lesions. It can be used to treat defects in epidermal adhesions. The polynucleotides or polypeptides of the invention, as well as agonists or antagonists, also treat gastric and duodenal ulcers and aid in scarring of the lining of the mucosa and faster healing by regeneration of the lining of the glandular and duodenal mucosa. Can be used for. Inflammatory bowel disease (
For example, Coulomb's disease and ulcerative colitis) are diseases that result in the disruption of the mucosal surface of the small or large intestine, respectively. Accordingly, the polynucleotides or polypeptides and / or agonists or antagonists of the present invention are intended to promote the resurfacing of mucosal surfaces to aid in faster healing and to prevent the progression of inflammatory bowel disease. Can be used. Treatment with the polynucleotides or polypeptides, agonists and / or antagonists of the invention is expected to have a significant effect on mucosal production throughout the gastrointestinal tract, and the intestinal mucosa is ingested or surgically operated. It can be used to protect against later harmful substances. The polynucleotides or polypeptides and / or agonists or antagonists of the invention can be used to treat diseases associated with the expression of the polynucleotides of the invention.

Furthermore, the polynucleotides or polypeptides and / or agonists or antagonists of the present invention can be used to prevent and cure damage to the lung due to various pathological conditions. The polynucleotides or polypeptides of the invention, and / or growth factors such as agonists or antagonists, can stimulate proliferation and differentiation to prevent or treat acute or chronic lung injury, and alveolar and bronchial ( Brochiolar) can promote epithelial repair. For example, emphysema (which results in progressive loss of alveoli) and inhalation injuries (ie, resulting from smoke inhalation and burns) resulting in necrosis of bronchial epithelium and aveoli of the present invention. It can be effectively treated using polynucleotides or polypeptides and / or agonists or antagonists. Also, the polynucleotides or polypeptides and / or agonists or antagonists of the invention can be used to stimulate alveolar epithelial cell type II proliferation and differentiation, which results in hyaline membrane disease in premature infants (eg, It can help treat or prevent diseases such as infant respiratory distress syndrome and bronchopulmonary dysplasia.

The polynucleotides or polypeptides and / or agonists or antagonists of the invention may stimulate the proliferation and differentiation of liver parenchymal cells, and thus liver diseases and conditions (eg fulminant liver failure caused by cirrhosis, hepatitis). Viral and toxic substances (ie acetaminophen, carbon tetrachloride (carbon)
It may be used to alleviate or treat liver damage) caused by tetrahollide), and other liver toxins known in the art).

In addition, the polynucleotides or polypeptides and / or agonists or antagonists of the invention can be used to treat or prevent the onset of diabetes mellitus. In patients newly diagnosed with type I and type II diabetes, if some islet cell function remains, the polynucleotides or polypeptides and / or agonists or antagonists of the present invention will not affect the persistence of the disease. It can be used to maintain its islet function so as to mitigate, delay or prevent expression. The polynucleotides or polypeptides of the invention, as well as agonists or antagonists, can also be used as an aid in islet cell transplantation to improve or promote islet cell function.

Neurological Disorders Nervous system diseases, disorders and / or that may be treated, prevented and / or diagnosed with the compositions (eg, polypeptides, polynucleotides and / or agonists or antagonists) of the invention. Conditions include nervous system damage and disease, which causes either axotomy, neuronal loss or degeneration, or demyelination.
Examples include, but are not limited to, disorders and / or conditions. Nervous system lesions that can be treated, prevented and / or diagnosed in patients (including human and non-human mammalian patients) according to the present invention include the following central nervous system (including spinal cord, brain)
Or any lesion of the peripheral nervous system, including but not limited to: (
1) ischemic lesions, where oxygen deficiency in a part of the nervous system causes neuronal damage or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia); (2) Traumatic lesions (including lesions that result from physical injury or are associated with surgery, such as lesions that cut through parts of the nervous system, or compression lesions)
(3) malignant lesion (where a part of the nervous system is destroyed or damaged by malignant tissue, which is either a nervous system-related malignant disease or a malignant disease derived from non-neural system tissue); (4) infectivity A lesion (where a portion of the nervous system is destroyed or damaged, for example, as a result of infection by an abscess, or is associated with infection by human immunodeficiency virus, herpes zoster or herpes simplex virus, Lyme disease, tuberculosis) , Associated with syphilis); (5) degenerative lesions, where parts of the nervous system are destroyed or damaged as a result of degenerative processes, including Parkinson's disease, Alzheimer's disease, Huntington's disease or the muscular atrophic side. (Including but not limited to degeneration associated with chord sclerosis (ALS)); (6) associated with nutritional diseases, disorders and / or conditions Lesions (where parts of the nervous system are destroyed or damaged by nutritional or metabolic disorders, including vitamin B12 deficiency, folate deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchia farva-Vignami disease (brain Primary degeneration of the corpus callosum and alcoholic cerebellar degeneration); (7) neurological lesions associated with systemic diseases (diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, cancer or the like). (8) lesions caused by toxic substances (including alcohol, lead or certain neurotoxins); and (9) demyelinating lesions (where a part of the nervous system is included). Is destroyed or damaged by demyelinating detention, which includes multiple sclerosis, human immunodeficiency virus-related myelopathy Transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and bridges but central myelinolysis include, but are not limited to).

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

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

In certain embodiments, a disease, disorder and / or condition of motor neurons that can be treated, prevented and / or diagnosed in accordance with the present invention is a disease that can affect motor neurons and other components of the nervous system, A disorder and / or condition (eg,
Infarction, infection, exposure to toxins, trauma, surgical injuries, degenerative or malignant disease),
And diseases, disorders and / or conditions that selectively affect neurons (eg, amyotrophic lateral sclerosis), and include, but are not limited to, progressive spinal muscular atrophy, Progressive bulbar palsy, primary lateral sclerosis, pediatric and juvenile muscular atrophy, childhood progressive bulbar palsy (Fatio-Ronde disease), polio and post-polio symptoms, and hereditary motor-sensory neuropathy (Charcot-Marie-Tooth disease), but is not limited thereto.

In addition, the polypeptides or polynucleotides of the present invention are useful for neuronal survival,
It may play a role in synapse formation; transmission; neural differentiation. Accordingly, the compositions of the present invention (including polynucleotides, polypeptides, and agonists or antagonists) are useful for diagnosing diseases or disorders associated with these roles, including but not limited to learning and / or cognitive disorders. , And / or can be used to treat or prevent. The compositions of the present invention may be useful in treating or preventing neurodegenerative disease states and / or behavioral disorders. Such neurodegenerative disease states and / or behavioral disorders include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Tourette's syndrome,
Schizophrenia, mania, dementia, paranoia, obsessive-compulsive disorder, depression, panic disorder, learning disability,
ALS, psychosis, autism, and behavioral changes (including impaired nutrition, sleep patterns, balance, and perception). In addition, the compositions of the invention may play a role in the treatment, prevention and / or detection of developing embryos, or developmental disorders associated with sexual disorders.

Further, the polypeptides, polynucleotides, and / or agonists or antagonists of the invention are from diseases, injuries, disorders or injuries associated with cerebrovascular disorders including, but not limited to: May be useful in protecting nerve cells: cerebrovascular disorders (eg, carotid thrombosis, carotid stenosis or Moyamoya disease), cerebral amyloid angiopathy, cerebral aneurysms, cerebral hypoxia, cerebral arteriosclerosis , Cerebral arteriovenous malformations, cerebral artery disease, cerebral embolism and thrombosis (eg carotid artery thrombosis, sinus thrombosis and Wallenberg syndrome), epidural hematoma (eg epidural or subdural hematoma and spider) Submembranous hemorrhage), cerebral fissure fracture, cerebral ischemia (eg,
Transient cerebral ischemia, subclavian steal syndrome, or vertebral basilar failure
basal insufficiency)), vascular dementia (eg, multiple cerebral infarction dementia), periventricular leukomalacia, and vascular headache (eg, cluster headache).

According to a still further aspect of the invention, for utilizing a polynucleotide or polypeptide of the invention and an agonist or antagonist for therapeutic purposes, eg to stimulate neurological cell proliferation and / or differentiation. Process is provided. Thus, the polynucleotides, polypeptides, agonists and / or antagonists of the invention can be used to treat and / or detect neurological disorders. In addition, the polynucleotides or polypeptides of the invention, or agonists or antagonists, can be used as markers or detection agents for certain nervous system diseases or injuries.

Examples of neurological disorders that may be treated or detected with the polynucleotides, polypeptides, agonists and / or antagonists of the invention include the following: brain disorders (eg of maternal phenylketonuria). Metabolic brain diseases including phenylketonuria), pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, Wernicke encephalopathy, cerebral edema, infratent (infr
cerebellar neoplasms such as cerebellar neoplasms including neoplasms, ventricular neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms, tentative neoplasms, Canavan's disease, ataxia-telangiectasia. Cerebellar diseases such as cerebellar ataxia including spinocerebellar ataxia, cerebellar dyskinesia, Friedreich's ataxia, Machado-Joseph disease, olive bridge cerebellar atrophy, cerebellar neoplasms such as subtentorial neoplasms Diffuse cerebral sclerosis, such as diffuse periaxial encephalitis, spheroid leukodystrophy, metachromatic leukodystrophy and subacute sclerosing panencephalitis.

The polynucleotides, polypeptides, agonists and / or antagonists of the invention can be treated or detected with as additional neurological disorders including:
These include: cerebrovascular disorders (eg, carotid artery thrombosis, carotid artery disease including carotid stenosis and Moyamoya disease), cerebral amyloid angiopathy, cerebral aneurysms, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebrum Cerebral embolism and thrombosis such as arterial disease, carotid thrombosis, sinus thrombosis and Wallenberg syndrome, cerebral hemorrhage such as epidural hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral fissure fracture, transient Cerebral ischemia, subclavian artery stealing syndrome and vertebrobasilar insuffi
cerebral ischemia such as ciency), vascular dementia such as multiple cerebral infarction dementia, periventricular leukomalacia, and vascular headache such as cluster headache.

Additional neurological disorders that may be treated or detected with the polynucleotides, polypeptides, agonists and / or antagonists of the invention include: dementia such as AIDS dementia, Alzheimer's disease and Creutzfeld-senile dementia such as Jacob's disease, senile dementia such as Alzheimer's disease and progressive supranuclear palsy, vascular dementia such as multiple cerebral infarction dementia, encephalitis such as diffuse periaxial encephalitis, Epidemic encephalitis, Japanese encephalitis, St. Louis encephalitis, tick-borne encephalitis and viral encephalitis such as West Nile fever, acute disseminated encephalomyelitis, uveal meningitis syndrome, post-encephalitis Parkinson's disease and subcytic sclerosis pancreatitis Meningoencephalomyelitis such as encephalitis, encephalomalacia such as periventricular leukoplakia, epilepsy such as generalized epilepsy including nasal spasm , Apusansu epilepsy (absence epilepsy), ME
Myoclonus epilepsy including RRF syndrome, ankylosis / clonic epilepsy, complex partial epilepsy, partial epilepsy such as frontal lobe epilepsy and temporal lobe epilepsy, post-traumatic status epilepticus such as continuous partial epilepsy, Hallerfolden -Spatz syndrome.

Additional neurological disorders that may be treated or detected with the polynucleotides, polypeptides, agonists and / or antagonists of the present invention include: Dandy-Walker syndrome and normal pressure hydrocephalus. Hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria, narcolepsy including weakness seizures, bulbar poomyelitis, cerebral pseudotumor, Rett's syndrome, Reye's syndrome, thalamic diseases, cerebral toxoplasmosis, cranium Internal tuberculoma and Zellweger syndrome, central nervous system infections such as AIDS dementia complex, brain abscess, subdural empyema, encephalomyelitis like equine encephalomyelitis, Venezuelan equine encephalomyelitis, necrotic hemorrhage Encephalomyelitis, visna, and cerebral malaria.

Additional neurological disorders that may be treated or detected with the polynucleotides, polypeptides, agonists and / or antagonists of the invention include: meningitis such as arachnoiditis, lymphocytes. Aseptic meningitis, such as viral meningitis, including congenital choroidal meningitis, bacterial meningitis, including Haemophilus meningitis, Listeria meningitis, meninges such as Waterhouse-Friedericksen syndrome Pneumococcal meningitis, pneumococcal meningitis and meningitis, fungal meningitis like cryptococcal meningitis, subdural exudation, meningoencephalitis like uveal meningoencephalitis syndrome ,
Myelitis such as transverse myelitis, neurosyphilis such as spinal fistula, poliomyelitis including medullary poliomyelitis and post-polio syndrome, prion diseases (eg Creutzfeld-Jakob disease, bovine spongiform encephalopathy, Gerstcon- (Stroisler syndrome, kuru, scrapie), as well as cerebral toxoplasmosis.

Additional neurological disorders that can be treated or detected with the polynucleotides, polypeptides, agonists and / or antagonists of the present invention include: cerebellar neoplasms such as subtentorial neoplasms. Spinal cord neoplasms including central nervous system neoplasms such as brain neoplasms, choroid plexus neoplasms, ventricular neoplasms such as hypothalamic neoplasms and supratentorial neoplasms, meningeal neoplasms, epidural neoplasms. Organisms, demyelinating diseases such as Canavan's disease, diffuse cerebral sclerosis including adrenoleukodystrophy (dif
fuse cerebral ceroris), diffuse periaxial encephalitis, bulbous cell leukodystrophy, metachromatic leukodystrophy diffuse cerebral sclerosis, allergic encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, progressive polymorphism Focal leukoencephalopathy, multiple sclerosis,
Hashi central myelinolysis, transverse myelitis, neuromyelitis optica, scrapie, lordosis, chronic fatigue syndrome, visna, high pressure nervous syndrome (High Pressure Ne)
rvous Syndrome), meningopathy, spinal cord diseases such as congenital myotonia, amyotrophic lateral sclerosis, spinal muscle atrophy such as Verdnig-Hoffmann disease, spinal cord squeezing, epidural neoplasia Spinal neoplasms, syringomyelia, spinal fistula, Stiffman's syndrome, mental retardation such as maternal 15q-13 microdeficiency, cat squealing syndrome,
De Lange syndrome, Down syndrome, gangliosidosis such as gangliosidosis G (M1), Zanthof's disease, Tay-Sachs disease, heart nap disease, homocystinuria, Lawrence-Moon-Beedle syndrome, Resch-Nyhan syndrome, Maple syrup disease, mucolipidosis such as fucose storage disease, neuronal ceroid lipofuscinosis, ocular brain kidney syndrome, phenylketonuria such as maternal phenylketonuria, Prader-Villi syndrome, Rett syndrome, Rubinstein- Nervous system abnormalities such as theaby's syndrome, tuberous sclerosis, WAGR syndrome, nervous system abnormalities such as panforencephalopathy, incompetence including hydrogenesis
Spinal cord fusion defects such as Arnold-Chiari malformations, cerebral hernias, meningocele, meningocele, cystic spina bifida and occult spina bifida.

Additional neurological disorders that may be treated or detected with the polynucleotides, polypeptides, agonists and / or antagonists of the present invention include: Hereditary motor and sensory disorders including Charcot-Marie disease. Neuropathy, visual atrophy, Refsum disease, hereditary spastic paraplegia, Verdnig-Hoffmann disease, hereditary sensory and autonomic neuropathy such as congenital analgesia and familial autonomic neuropathy, neurological manifestations (Eg, agnosia including Gerstmann syndrome), amnesia such as retrograde amnesia, apraxia, neurogenic bladder, weakness, deafness,
Communication impairments such as hearing loss including partial hearing loss, ludness recruitment and tinnitus, aphasia, speech disorders such as aphasia, aphasia including Broka aphasia and Wernicke aphasia, acute dyslexia Communication such as dyslexia, language development disorders, name aphasia, aphasia including aphasia including Broca aphasia and Wernicke aphasia, accumulative disorders, dyslexia, reverberant language, verbal disorders including speechlessness and stuttering Disorders, dysphonia such as aphonia and hoarseness, decerebrate rigidity, delirium, fasciculations, hallucinations, meningopathy, 15q-13 microdefects from mother, ataxia, athetosis, chorea, ataxia, Like hypokinesis, hypotonia, movement disorders like myoclonus, tics, torticollis and tremors, muscle stiffness like Stiffman syndrome Muscular hypertonia, muscular spasticity, nerve palsy such as facial palsy including ear zoster, gastroparesis, hemiplegia, ophthalmoplegia such as diplopia, chronic such as duene syndrome, horner syndrome, keens syndrome Of paraplegia, paresis, phantom limbs, dysgeusia and dysgeusia such as progressive extraocular muscle palsy, bulbar palsy, tropical spastic paraparesis, Brown-Secar syndrome, quadriplegia, respiratory palsy and vocal cord paralysis Dysgeusia, amblyopia, blindness, color blindness, diplopia, semi-blindness, visual disorders such as scotoma and subnormal vision, Kleine-Levin syndrome, hypersomnia including insomnia and sleepwalking Sleep disorders such as, spasticity such as trismus, coma, persistent vegetative state and loss of consciousness such as syncope and dizziness, neuromuscular disorders such as congenital myotonia, muscular atrophic side Cord sclerosis, Lambert-Eaton myasthenia syndrome, motor neuron disease, spinal muscle atrophy, muscle atrophy such as Charcot-Marie disease and Verdnig-Hoffmann disease, post-polio syndrome, muscular dystrophy, myasthenia gravis, atrophic muscle Tension, congenital myotonia, nemarin myopathy, familial quadriplegia, multiple paramyloclonus (Multiplex Paramyl)
oclonus), tropical spasmodic paraplegia and Stiffman syndrome, peripheral nervous system diseases such as acroampalgia, renal amyloidosis, Ardy syndrome, B
such as arre-Lieou syndrome, familial autonomic neuropathy, Horner syndrome, autonomic nervous system diseases such as reflex sympathetic dystrophy and Shy-Drager syndrome, inner ear nerve diseases such as acoustic neuroma including neurofibromatosis 2. Cranial nerve disease,
Facial nerve diseases such as facial neuralgia, Merkerson-Rosenthal syndrome, ocular motility disorders including amblyopia, nystagmus, oculomotor paralysis, ophthalmoplegia such as Duane's syndrome, horner syndrome, chronic progressive disease including Keynes syndrome External ophthalmoplegia, strabismus such as strabismus and exotropia, oculomotor nerve palsy, optic nerve diseases such as ocular atrophy including inherited optic atrophy, optic disc disk, optic nerve such as optic myelitis Inflammation, papilledema,
Demyelinating diseases such as trigeminal neuralgia, vocal cord paralysis, neuromyelitis optica and lordosis, diabetic neuropathy such as diabetic foot.

Additional neurological disorders that may be treated or detected with the polynucleotides, polypeptides, agonists and / or antagonists of the invention include: nerve squeezing syndrome, such as carpal tunnel syndrome, foot Root canal syndrome, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve squeeze syndrome, causalgia, brachial neuralgia,
Neuralgias such as facial neuralgia and trigeminal neuralgia, experimental allergic neuritis, optic neuritis, polyneuritis, polyradiculoneuritis and radiculitis (eg polyradiculitis, hereditary movements and sensations) Neuropathies (eg, Charcot-Marie disease, hereditary eye atrophy, Refsum disease, hereditary spastic paraplegia and Verdnig-Hoffmann disease, hereditary sensory and autonomic neuropathy (congenital analgesia and familial autonomic neuropathy). , POEMS syndrome, sciatica, taste sweating syndrome and tetany)).

Infectious Diseases The polynucleotides or polypeptides, and / or agonists or antagonists of the invention can be used to treat, prevent and / or diagnose infectious agents. For example, infectious diseases can be treated, prevented, and / or diagnosed by raising an immune response, particularly by increasing B cell and / or T cell proliferation and differentiation. The immune response can be elevated by either raising an existing immune response or initiating a new immune response. Alternatively, the polynucleotides or polypeptides of the invention and / or agonists or antagonists also do not necessarily elicit an immune response,
Infectious agents can be directly inhibited.

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

Similarly, bacterial or fungal agents which can cause a disease or condition and which can be treated, prevented and / or diagnosed by the polynucleotides or polypeptides of the invention and / or agonists or antagonists are: Includes, but is not limited to, Gram-negative and Gram-positive bacteria and bacteria and fungi: Actinomycetales (eg, Corynebac
terium, Mycobacterium, Norcardia), Cryp
tococcus neoformans, Aspergillosis, Ba
cillaceae (eg, Anthrax, Clostridium), B
acteroidaceae, Blastomycosis, Bordetel
la, Borrelia (eg, Borrelia burgdorferi
), Brucellosis, Candidiasis, Campylobacter
ter, Coccidioidomycosis, Cryptococcosi
S., Dermatocycleses, E.I. coli (for example, Enteroto
xigenic E. E. coli and Enterohemorrhagic E
． E. coli), Enterobacteriaceae (Klebsiella)
, Salmonella (eg, Salmonella typhi, and Salmonella paratyphi), Serratia, Yersi.
nia), Erysipelothrix, Helicobacter, Leg
ionellosis, Leptospirosis, Listeria, My
coplasmatales, Mycobacterium leprae, V
ibrio cholerae, Neisseriaceae (eg Aci
netobacter, Gonorrhea, Menigococcal), M
eisseria meningitidis, Pasteurellacea
Infectious diseases (for example, Actinobacillus, Hemophilus (
For example, Hemophilus influenza B), Pasteurell
a), Pseudomonas, Rickettsiaceae, Chlamy
diaceae, Syphilis, Shigella spp. , Staph
ylococcal, Meningiococcal, Pneumococca
l and Streptococcal (eg, Streptococcus
Pneumoniae and Group B Streptococcus). These bacterial or fungal families can cause diseases or conditions including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunism. Infections (eg, AIDS-related infections), periungual inflammation, prosthesis-related infections, Reiter's disease, respiratory tract infections (eg, pertussis or pyometra), sepsis, Lyme disease, cat scratch disease, dysentery, Paratyphoid fever, Food poisoning, typhoid fever, pneumonia, gonorrhea, meningitis (eg meningitis type A and B), chlamydia, syphilis, diphtheria, leprosy, paratuberculosis, tuberculosis, lupus, botulinum poisoning, gangrene, tetanus, impetigo , Rheumatic fever, scarlet fever, sexually transmitted diseases, skin diseases (eg cellulitis, dermatomycosis (de)
rmatocycles)), toxemia, urinary tract infection, wound infection. The polypeptides or polynucleotides, agonists or antagonists of the invention may be used to treat, prevent and / or diagnose any of these conditions or diseases. In certain embodiments, the polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat, prevent and / or diagnose the following: tetanus, diphtheria, botulinum, and / or meningitis type B. .

Furthermore, diseases or conditions caused by parasitic factors that can be treated, prevented and / or diagnosed with the polynucleotides or polypeptides of the invention and / or agonists or antagonists include the following families or classes: Are not limited to these: amebiasis, babesiosis, coccidiosis, cryptosporidiosis, dinuclear amebiosis (Dientamoebi)
as), copulation, ectoparasite, giardiasis, helminthosis, leishmaniasis, theileriosis, toxoplasmosis, trypanosomiasis, and Trichomonas disease, and sporozoan disease (Sporozoan).
(For example, Plasmodium virax, Plasmodium fal
ciparium, Plasmodium malariae and Plasm
odium ovale). These parasites can cause a variety of diseases or conditions including, but not limited to: scabies, scrub typhus, ocular infections, intestinal diseases (eg, dysentery, giardia giardiasis), liver diseases, lungs. Diseases, opportunistic infections (eg, AIDS-related), malaria, pregnancy complications, and toxoplasmosis. The polynucleotides or polypeptides of the invention, or agonists or antagonists, may be used to treat, prevent and / or diagnose any of these conditions or diseases. In a particular embodiment, the polynucleotide of the invention,
The polypeptide, or agonist or antagonist treats malaria,
Used for prevention and / or diagnosis.

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

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

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

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

Similarly, neural 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 neural cells. Treatment using this method,
Diseases that can be prevented and / or diagnosed include central nervous system diseases and peripheral nervous system diseases, neuropathy, or mechanical and traumatic diseases, disorders and / or conditions (for example, spinal cord disorders, head trauma, cerebrovascular diseases). Diseases, and strokes). In particular, diseases associated with peripheral nerve injury, peripheral neuropathy (eg resulting from chemotherapy or other medical therapies), localized neuropathy, and central nervous system diseases (eg Alzheimer's disease, Parkinson's disease, Huntington). Disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome) can all be treated, prevented and / or diagnosed with the polynucleotides or polypeptides of the invention and / or agonists or antagonists.

Chemotaxis The polynucleotides or polypeptides and / or agonists or antagonists of the invention may have chemotactic activity. Chemotactic molecules allow cells (eg, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells and / or endothelial cells) to move to specific sites in the body (eg, Site of inflammation, infection, or hyperproliferation). The recruited cells may then repel and / or heal the particular trauma or abnormality.

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

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

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

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

Preferably, screening for these molecules involves producing suitable cells that express the polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include mammals, yeast, Drosoph
ila, or E.I. cells derived from E. coli. The cells expressing the polypeptide (or cell membranes containing the expressed polypeptide) are then preferably observed for binding, stimulating, or inhibiting activity of either the polypeptide or the molecule. Are contacted with a test compound potentially containing the molecule of

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

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

[0560] Preferably, the ELISA assay may measure the level or activity of a polypeptide in a sample (eg, biological sample) using monoclonal or polyclonal antibodies. Antibodies may be bound to the polypeptide either directly or indirectly, or by competition with the polypeptide for a substrate,
The level or activity of the polypeptide can be measured.

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

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

As an alternative approach for receptor identification, labeled polypeptides may be photoaffinity-linked or extracted with a preparation expressing the receptor molecule, photoaffinity. The crosslinked material is separated by PAGE analysis and exposed to X-ray film. A labeled complex containing the receptor for the polypeptide can be excised,
It can be separated into peptide fragments and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing is used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.

Furthermore, the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as “DNA shuffling”) may be used to modulate the activity of the polypeptides of the invention, thereby. Effectively produces agonists and antagonists of the polypeptides of the invention. Generally, US Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,83.
7,458, and Patten, P .; A. Curr. Opinion
Biotechnol. 8: 724-33 (1997); Harayama,
S. Trends Biotechnol. 16 (2): 76-82 (1998)
); Hansson, L .; O. Et al., J. Mol. Biol. 287: 265-7
6 (1999); and Lorenzo, M .; M. And Blasco, R .;
See Biotechniques 24 (2): 308-13 (1998), each of which patents and publications are incorporated herein by reference. In one embodiment, alteration of the polynucleotides of the invention and corresponding polypeptides can be accomplished by DNA shuffling. DNA shuffling is the process of homologous recombination or site-specific recombination of two or more DNA fragments.
Including the construction of the segment into the desired polynucleotide sequence of the molecule of the present invention. In another embodiment, the polynucleotides of the invention and the corresponding polypeptides are subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. Can be changed. In another embodiment, one or more components of the polypeptide of the invention,
A motif, segment, portion, domain, fragment, etc. can be recombined with one or more components, motifs, segments, portions, domains, fragments, etc. of one or more heterologous molecules. In a preferred embodiment, the heterologous molecule is a member of the family. In a further preferred embodiment, the heterologous molecule is, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF) -α, epidermal growth factor (EGF), fibroblast. Cell growth factor (FGF), TGF-β, bone morphogenetic protein (BMP) -2, BMP-4, BM
P-5, BMP-6, BMP-7, activin A and activin B, decapentaplegic (dpp), 60A, OP-
2, dosalin, growth differentiation factor (GDF), nodule (nod)
al), MIS, inhibin-α, TGF-β1, TGF-β2, TGF-β3.
, TGF-β5, and growth factors such as glial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of the polypeptides of the invention. A biologically active fragment is a fragment that exhibits an activity similar to, but not necessarily identical to, the activity of this polypeptide. The biological activity of this fragment may include an improved desired activity, or a decreased undesired activity.

The present invention further provides methods of screening compounds to identify those that modulate the action of the polypeptides of the present invention. An example of such an assay involves combining mammalian fibroblasts, a polypeptide of the invention, the compound to be screened and ³ [H] thymidine under cell culture conditions in which the fibroblasts normally grow. . The control assay can be carried out in the absence of the compound to be screened, and compared to the amount of proliferation of fibroblasts in the presence of this compound, in each case of ³ [H] thymidine incorporation. The determination can determine whether the compound stimulates proliferation. The amount of fibroblast proliferation is ³ [
Measured by liquid scintillation chromatography, which measures [H] thymidine incorporation. 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 and receptor of a known second messenger system according to the interaction of the compound to be screened is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to Determine if it is a potential agonist or antagonist. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognostic markers. Molecules discovered using these assays treat, prevent, and / or diagnose a disease by activating or inhibiting a polypeptide / molecule, or have a patient-specific outcome (eg, blood vessel growth). Can be used to bring. In addition, the assay can discover factors that can inhibit or enhance production of the polypeptides of the invention from appropriately 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 a candidate binding compound with the polypeptide; and (b) whether binding has occurred. The process of determining whether or not. Further, the invention includes a method of identifying an agonist / antagonist comprising the steps of: (a) incubating a candidate compound with a polypeptide, (b) assaying biological activity, and (b) ) Determining whether the biological activity of the polypeptide has been modified.

Also, the β-pleated sheet region contained in the polypeptide sequence of a protein can be used to experimentally identify molecules that bind the polypeptide of the invention. Accordingly, certain embodiments of the invention include the amino acid sequence of each β-pleated sheet region in the disclosed polypeptide sequences, or alternatively, the amino acid sequence of each β-pleated sheet region in the disclosed polypeptide sequences. Consisting of a polynucleotide encoding a polypeptide. A further embodiment of the invention encodes a polypeptide comprising any combination or all contained in a polypeptide sequence of the invention, or consisting of any combination or all contained in a polypeptide sequence of the invention. To a polynucleotide. A further preferred embodiment of the invention comprises the amino acid sequence of each of the β pleated sheet regions in one of the polypeptide sequences of the invention, or β in one of the polypeptide sequences of the invention.
It relates to a polypeptide consisting of the amino acid sequence of each of the pleated sheet regions. Further embodiments of the invention include any combination or all of the β-pleated sheet regions in one of the polypeptide sequences of the invention, or any of the β-pleated sheet regions in one of the polypeptide sequences of the invention. It relates to polypeptides which consist of combinations or all.

Targeted Delivery In another embodiment, the invention provides a composition comprising a targeted cell expressing a receptor for a polypeptide of the invention, or a cell-bound form of a polypeptide of the invention. Methods of delivering to expressing cells are provided.

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

In another embodiment, the invention provides cell specificity by administering a polypeptide of the invention (eg, a polypeptide of the invention or an antibody of the invention) associated with a toxin or a cytotoxic prodrug. Methods for selective destruction (eg, destruction of tumor cells).

A “toxin” is an endogenous cytotoxic effector system, radioisotope, holotoxin, modified toxin, catalytic subunit of a toxin, or a cell that causes cell death under defined conditions. Alternatively, it means a compound that binds and activates any molecule or enzyme not normally present on the cell surface. Toxins that may be used in accordance with the methods of the present invention include, but are not limited to, radioisotopes known in the art, compounds that bind an intrinsic or induced endogenous cytotoxic effector system. (For example, antibody (or complement fixation including a part thereof), thymidine kinase, endonuclease, RNAse, α-toxin, ricin,
Abrin, Pseudomonas endotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. "
By "cytotoxic prodrug" is meant a non-toxic compound that is converted into a cytotoxic compound by the enzymes normally present in cells. Cytotoxic prodrugs that may 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. However, the present invention is not limited to these.

Drug Screening Further contemplated is the use of the polypeptides of the invention, or polynucleotides encoding these polypeptides, to screen for molecules that modify the activity of the polypeptides of the invention. Such methods include the steps of contacting the polypeptides of the invention with selected compounds suspected of having antagonistic or agonistic activity, and assaying the activity of these polypeptides following binding. To do.

The present invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment used 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 which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. For example, the formation of a complex between the agent being tested and the polypeptide of the invention can be measured.

Accordingly, the invention provides methods of screening for drugs or any other agents that affect the activity mediated by the polypeptides of the invention. These methods involve contacting such agents with a polypeptide of the invention or fragment thereof by methods well known in the art, and the presence of a complex between the agent and the polypeptide or fragment thereof. The step of assaying is included. In such competitive binding assays, the drug to be screened is typically labeled. After 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 drug to bind a polypeptide of the invention. .

Another technique for drug screening provides high throughput screening for compounds with appropriate binding affinity for the polypeptides of the invention, and European Patent Application 84/03564 (published September 13, 1984). (Which is incorporated herein by reference) in great detail. Briefly, large numbers of different small peptide test compounds are synthesized on a solid substrate (eg, plastic pins or some other surface). Peptide test compound,
React with polypeptide of the invention and wash. Bound polypeptide is then detected by methods well known in the art. The purified polypeptide is coded directly on the plate for use in the drug screening techniques described above. 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 a neutralizing antibody capable of binding a polypeptide of the invention specifically competes with a test compound for binding to the polypeptide or fragment thereof. To 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.

Peptides and Other Molecules that Bind to the Polypeptides of the Invention The invention also features screening methods for identifying polypeptides and non-polypeptides that bind to the polypeptides of the invention, and thereby identified. The polypeptide of the invention also comprises a binding molecule. These binding molecules are useful, for example, as agonists and antagonists of the polypeptides of the invention. Such agonists and antagonists can be used in accordance with the present invention in the therapeutic embodiments described in detail below.

[0580]   The method comprises the following steps:   a. Contacting the polypeptide of the invention with a number of molecules; and   b. Identifying a molecule that binds to a polypeptide of the invention.

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

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

In certain circumstances, prior to attempting to determine or detect the presence of a selective affinity interaction, either unbound polypeptide of the present invention or unbound polypeptide of the present invention and polypeptide of the present invention It may be desirable to wash away a mixture of multiple polypeptides. Such washing steps may be particularly desirable when the polypeptide of the invention or a large number of polypeptides are bound to a solid support.

A large number of molecules provided in accordance with the present method can be used in diversity libraries (eg, random or combinatorial peptide libraries or non-peptide live libraries that can be screened for molecules that specifically bind to the polypeptides of the invention). Rally). Many libraries that can be used (eg, chemical synthesis libraries, recombinant libraries (eg, phage display libraries),
And in vitro translation-based libraries) are known in the art. Examples of chemically synthesized libraries are described below: Fodor et al., 1991, Sci.
ence 251: 767-773; Houghten et al., 1991, Natu.
Re 354: 84-86; Lam et al., 1991, Nature 354: 82.
-84; Medynski, 1994, Bio / Technology 12:
709-710; Gallop et al., 1994, J. Am. Medicinal Che
mistry 37 (9): 1233-1251; Ohlmeyer et al., 199.
3, Proc. Natl. Acad. Sci. USA 90: 10922-10.
926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA
91: 11422-11426; Houghten et al., 1992, Biote.
chniques 13: 412; Jaywickreme et al., 1994, P.
roc. Natl. Acad. Sci. USA 91: 1614-1618; S.
almon et al., 1993, Proc. Natl. Acad. Sci. USA 9
0: 11708-11712; PCT Publication No. WO93 / 20242; and Brenner and Lerner, 1992, Proc. Natl. Acad
． Sci. USA 89: 5381-5383.

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

In vitro translation-based libraries include: PCT Publication No. WO9
1/05058 (April 18, 1991); and Mattheakis et al., 1
994, Proc. Natl. Acad. Sci. USA 91: 9022-9.
026, but is not limited thereto.

As an example of a non-peptide library, a benzodiazepine library (eg, Bunin et al., 1994, Proc. Natl. Acad. Sci. USA).
91: 4708-4712) can be adapted for use. Peptoid library (Simon et al., 1992, Proc. Natl. Acad
． Sci. USA 89: 9367-9371) may also be used. Another example of a library that can be used is Ostresh et al. (1994, Proc. Nat.
l. Acad. Sci. USA 91: 11138-11142), in which the amide functional group in the peptide is hypermethylated.
to produce a chemically transformed combinatorial library.

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

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

Non-peptidic oligomer libraries use a large number of monomers that are assembled together in a manner that creates new shapes depending on the order of the monomers. Among the monomer units used are carbamates, pyrrolinones.
) And morpholino. Peptoids, peptide-like oligomers whose side chains bind to α-amino groups rather than α-carbons, form the basis of another version of non-peptide oligomer libraries. The first non-peptide oligomer library uses a single type of monomer and thus contains a repeating backbone. Modern libraries use more than one monomer, giving libraries with added degrees of freedom.

Screening the library can be accomplished by any of a variety of commonly known methods. For example, the following references disclosing the screening of peptide libraries: Parmley and Smith, 1989, Adv.
． Exp. Med. Biol. 251: 215-218; Scott and Sm.
ith, 1990, Science 249: 386-390; Fowlkes.
Et al., 1992; BioTechniques 13: 422-427; Olde.
nburg et al., 1992, Proc. Natl. Acad. Sci. USA 8
9: 5393-5397; Yu et al., 1994, Cell 76: 933-945.
Staudt et al., 1988, Science 241: 577-580; Bo.
ck et al., 1992, Nature 355: 564-566; Turerk et al., 1.
992, Proc. Natl. Acad. Sci. USA 89: 6988-6.
992; Ellington et al., 1992, Nature 355: 850-8.
52; US Pat. No. 5,096,815, US Pat. No. 5,223,409, and US Pat. No. 5,198,346 (all to Ladner et al.); Reb.
See ar and Pabo, 1993, Science 263: 671-673; and PCT Publication No. WO94 / 18318.

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

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

When the polypeptide of the present invention bound to a molecule is a polypeptide, the polypeptide can be any peptide library (random peptide library,
(Including combinatorial peptide libraries, or biased peptide libraries). The term “biased” means in this case, the method by which the library is made in the peptide is engineered to limit one or more parameters that govern the diversity of the resulting molecular collection. As used herein.

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

As noted above, a molecule (which is a polypeptide) that binds to a polypeptide of the invention.
Wherein the polypeptide has from about 6 to less than about 60 amino acid residues,
It can be preferably about 6 to about 10 amino acid residues, and more preferably about 6 to about 22 amino acids. In another embodiment, the polypeptide that binds to the polypeptide of the invention has amino acids in the range 15-100, or amino acids in the range 20-50.

A selected polypeptide of the invention attached to a molecule may be obtained by chemical synthesis or recombinant expression.

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

For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines has been previously described (Wickstrom et al. 1988); Anfossi et al.
9)). These experiments were performed in vitro by incubating cells with oligoribonucleotides. A similar procedure for in vivo use is
It is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is generated as follows: a sequence complementary to the first 15 bases of the open reading frame, an EcoRI site at the 5'end and a HindIII at the 3'end. Adjacent to the site. The oligonucleotide pairs are then heated at 90 ° C. for 1 minute and then 2 × ligation buffer (20 mM TRIS HC
pH 7.5, 10 mM MgCl ₂ , 10 mM dithiothreitol (DT
T) and 0.2 mM ATP) and then ligated into the EcoR1 / HindIII sites of the retroviral vector PMV7 (WO91).
/ 15580).

For example, the 5'coding portion of the polynucleotide encoding the mature polypeptide of the invention can be used to design an antisense RNA oligonucleotide of about 10-40 base pairs in length. DNA oligonucleotides are designed to be complementary to regions of the gene involved in transcription, thereby inhibiting transcription and production of receptors. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into a receptor polypeptide.

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 to produce the antisense nucleic acid (RNA) of the invention. Such a vector contains a sequence encoding the antisense nucleic acid of the present invention. Such a vector can be episomal or chromosomally integrated, 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. The vector can be a plasmid, virus, etc. known in the art used for replication and expression in vertebrate cells. Expression of sequences encoding the polypeptides of the present invention or fragments thereof may be obtained by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include the SV40 early promoter region (Bernist and Chambon, Nature, 29: 304-3.
10 (1981)), a promoter contained in the 3'-long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22: 787-797 (1980)),
Herpes thymidine promoter (Wagner et al., Proc. Natl. Aca
d. Sci. U. S. A. 78: 1441-1445 (1981)), regulatory sequences of the metallothionein gene (Brinster et al., Nature, 296: 39).
-42 (1982)) and the like, but are not limited thereto.

The antisense nucleic acid of the present invention contains a sequence complementary to at least a part of RNA transcript of the gene of interest. However, perfect complementarity, although preferred, is not necessary. A sequence "complementary to at least a portion of RNA" as referred to herein means a sequence that has sufficient complementarity to hybridize with RNA and forms a stable duplex; Thus, in the case of the double-stranded antisense nucleic acids of the invention, single strands of double-stranded DNA can be tested or triplex formation can be assayed.
The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid. In general, longer hybridizing nucleic acids may contain more base mismatches with the RNA sequences of the invention, which may include stable duplexes (or possibly triplexes), And yet it can be formed. One of skill in the art can ascertain the degree of mismatch tolerance by using standard procedures to determine the melting temperature of the hybridizing complex.

Oligonucleotides that are complementary to the 5'end of the message (eg, 5'untranslated sequences up to and including the AUG start codon) should work most efficiently in inhibiting translation. is there. However, sequences complementary to the 3'untranslated sequences of mRNAs have likewise been shown to be effective in inhibiting translation of mRNAs. Generally, Wagner, R .; , Nature 372: 333-335 (199).
See 4). Therefore, 5'- or 3'of the polynucleotide sequence of the present invention
Oligonucleotides complementary to any of the untranslated non-coding regions of
It can be used in an antisense approach that inhibits translation of RNA. 5 of mRNA
The oligonucleotide complementary to the'untranslated region should include the complement of the AUG start codon. Antisense oligonucleotides complementary to the mRNA coding region, although less efficient inhibitors of translation, can be used in accordance with the present invention. Whether designed to hybridize to the 5'region, 3'region, or coding region of the mRNA, the antisense nucleic acid should be at least 6 nucleotides in length, and preferably 6 to about 50. An oligonucleotide that spans the length of a nucleotide. In certain aspects, the oligonucleotide is 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, or derivative or modified versions thereof, single-stranded, or double-stranded. The oligonucleotide may be modified at the base moiety, sugar moiety, or phosphate backbone to improve, eg, molecular stability, hybridization, and the like.
The oligonucleotide may be attached to other additional groups such as peptides (eg, for targeting 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); Lemait.
Re et al., Proc. Natl. Acad. Sci. 84: 648-652 (19
87); PCT Publication No. WO88 / 09810 (Published December 15, 1988)
, Or the blood-brain barrier (eg PCT Publication No. WO89 / 101).
34 (published April 25, 1988)), hybridization-triggered cleavag.
e.g.) (eg Kroll et al., BioTechniques, 6: 9).
58-976 (1988)), or intercalating agents (see, for example, Zon, Pharm. Res. 5: 539-549 (1988)). For this purpose, the oligonucleotide is a different molecule (for example,
Peptides, hybridization-triggered cross-linking agents, transport agents, hybridization-triggered cleavage agents, etc.).

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

Antisense oligonucleotides may 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, phosphoro. Amidothioates, phosphoramidates, phosphorodiamidates, methylphosphonates, alkyl phosphotriesters, and formacetals or analogs thereof.

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

Polynucleotides of the invention may be prepared using standard methods known in the art (eg automated D
By NA synthesizer (such equipment is Biosearch, Applied B
(commercially available from iosystems, etc.)). For example, phosphorothioate oligonucleotides are described by Stein et al. (Nuc
l. Acids Res. 16: 3209 (1988)),
Methylphosphonate oligonucleotides are used as controlled pore glass (con
polymerized polymer support (Sarin et al., Pr)
oc. Natl. Acad. Sci. U. S. A. , 85: 7448-7451
(1988)) and the like.

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

Potential antagonists according to the present invention also include catalytic RNA, ie ribozymes (eg PCT International Publication WO 90/11364, October 4, 1990).
Published: Sarver et al., Science, 247: 1222-1225 (19).
90)). On the other hand, a ribozyme that cleaves an mRNA at a site-specific recognition sequence can be used to destroy the mRNA corresponding to the polynucleotide of the present invention, but it is preferable to use a hammerhead ribozyme. Hammerhead ribozymes cleave mRNAs at positions determined by flanking regions that form complementary base pairs with the target mRNA. The only requirement is that the target mRNA has the following two base sequences: 5'-UG-3 '. Construction and production of hammerhead ribozymes is well known in the art, and Haseloff and Ge
Rlach, 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 a cleavage recognition site is located near the 5'end of the mRNA corresponding to the polynucleotide of the invention; ie, it increases efficiency and promotes intracellular accumulation of non-functional mRNA transcripts. Manipulated to minimize.

In the case of the antisense approach, ribozymes of the invention may be composed of modified oligonucleotides (improved for stability, targeting, etc.) and express polynucleotides of the invention in vivo. Should be delivered to cells. The DNA construct encoding the ribozyme can be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery is to "encode" the ribozyme under the control of a strong constitutive promoter (such as the pol III or pol II promoters).
Including the use of the DNA construct so that the transfected cells disrupt the endogenous message and produce a sufficient amount of ribozyme to inhibit translation. Ribozymes, unlike antisense molecules, are catalytic, so lower intracellular concentrations are required for efficiency.

Antagonist / agonist compounds are utilized to grow and proliferate the polypeptides of the invention to neoplastic cells and tissues.
feration) effect. That is, stimulating a tumor agonist,
It slows or prevents aberrant cell growth and proliferation (eg, in tumorigenesis or proliferation).

Antagonists / agonists may also be utilized 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 in cases such as restenosis after balloon angioplasty.

Antagonists / agonists may also be utilized to prevent scar tissue growth during wound healing.

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

Accordingly, the present invention includes diseases, disorders and / or conditions (including diseases, disorders and / or conditions listed throughout this application which relate to overexpression of a polynucleotide of the invention, (Not limited to these), the method comprising: (a) an antisense molecule related to the polynucleotide of the present invention, and / or (b) a ribozyme related to the polynucleotide of the present invention. Or provide a method of prevention. The invention, and / or (b) a ribozyme relating to the polynucleotide of the present invention.

Other Activities The polypeptides of the present invention are responsible for various disease states (eg, thrombosis, arteriosclerosis, and other cardiovascular conditions) as a result of their ability to stimulate vascular endothelial cell proliferation. Can be used in a procedure for stimulating the revascularization of ischemic tissue. These polypeptides may also be utilized to stimulate angiogenesis and limb remodeling as discussed above.

The polypeptide may also be utilized in the treatment of wounds resulting from injury, burns, post-operative tissue repair, and ulcers. Because they are mitogenic for a variety of cells of different origin, such as fibroblasts and skeletal muscle cells, and therefore facilitate 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 such as Alzheimer's disease, Parkinson's disease, and AIDS-related complications. , Can be used for prevention and / or diagnosis. 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 grafts or bone grafts. .

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

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

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

The polypeptides of the present invention can also be utilized to induce tissues 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 regulate mammalian metabolism that affects catabolism, anabolic activity, processing, utilization, and energy storage.

Polypeptides or polynucleotides and / or agonists or antagonists of the invention may be used in biorhythms, caricadic
) Rhythm, depression (including depressive diseases, disorders and / or conditions), tendency to violence, resistance to pain, fertility (preferably by activin or inhibin-like activity), hormonal or endocrine levels, appetite , Can be used to alter a mammal's mental or physical condition by affecting libido, memory, stress, or other cognitive qualities.

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

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

The sequence of contiguous nucleotides begins at the nucleotide approximately 5'nucleotides of the clone sequence, as defined for SEQ ID NO: X in Table 1,
Also preferred is a nucleic acid molecule comprised in the nucleotide sequence of SEQ ID NO: X, in the range of positions ending with nucleotides at approximately the 3'nucleotide position of the clone sequence.

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

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

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

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

A further preferred embodiment is as set forth for SEQ ID NO: X in Table 1,
Includes a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: X that begins approximately at the 5'nucleotide position of the first amino acid of the signal peptide and ends approximately at the 3'nucleotide position of the clone sequence. It is a nucleic acid molecule.

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

Also preferred is an isolated nucleic acid molecule that hybridizes to a nucleic acid molecule under stringent hybridization conditions, wherein the hybridizing nucleic acid molecule above is an A residue under stringent hybridization conditions. Alone or does not hybridize to a nucleic acid molecule having a nucleotide sequence consisting of only 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 the material deposited with the American Type Culture Collection, and The ATCC deposit numbers shown in Table 1 are given for the above cDNA clone IDs.

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

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

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

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

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

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

Comparing the sequences described above includes 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. The above method is also preferable. Similarly, the step of comparing the sequences described above, with a nucleotide sequence determined from the nucleic acid molecules in the sample,
Also preferred is the above method carried out by the step of comparing with said sequence selected from the above group. Nucleic acid molecules can include DNA or RNA molecules.

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

The method for identifying the 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, where At least one sequence in the panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in the sequence selected from the group above.

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

Also preferred is a composition of matter comprising an isolated nucleic acid molecule, wherein the nucleotide sequence of said nucleic acid molecule comprises a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is , At least 95% identical to a 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 contained in the deposit identified by the cDNA Clone ID in Table 1 and having the ATCC Deposit Number shown for the above cDNA clone in Table 1. Nucleic acid molecules can include DNA or RNA molecules.

Also preferred is an isolated polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least about 10 consecutive amino acids in the amino acid sequence of SEQ ID NO: Y, where Y is in Table 1 It is an arbitrary integer as specified.

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

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

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

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

The cDNA clones identified by the cDNA clone ID in Table 1 and above in Table 1
Human c contained in the deposit with the ATCC deposit number indicated for the A clone
Further preferred is an isolated polypeptide comprising an amino acid sequence that is at least 90% identical to a 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 sequence of contiguous amino acids above is identified by the cDNA clone ID in Table 1 and encoded by the human cDNA clone contained in the deposit with the ATCC accession number shown for the above cDNA clone in Table 1. Also preferred is a polypeptide comprised in the amino acid sequence of the secretory portion of a secreted protein.

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

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

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

An isolated that specifically binds to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 consecutive amino acids in a sequence selected from the group consisting of: The antibody is 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. The complete amino acid sequence of the protein encoded by the human cDNA clone contained in the deposit with the indicated ATCC Deposit Number.

A method of detecting in a biological sample a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 consecutive amino acids in a sequence selected from the group consisting of: Preferred: amino acid sequence of SEQ ID NO: Y, where Y is any integer 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. 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 this sample to a sequence selected from this group. The process and distribution of this polypeptide molecule in this sample. A row contains at least 9 sequences of this sequence of at least 10 consecutive amino acids.
Includes determining if 0% identity.

This step of comparing the amino acid sequence of at least one polypeptide molecule in this sample with a sequence selected from this group is carried out in a sequence of polypeptides of this sample 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 consecutive 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 indicated for this cDNA clone in Table 1.
The complete amino acid sequence of the protein encoded by the human cDNA clone contained in the deposit with the C accession number.

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

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

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. At least one sequence in this panel is included in at least one of the sequences selected from the group above.
At least 90% identical to a sequence of 0 consecutive amino acids.

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

In any of these methods, the step of detecting the polypeptide molecule comprises:
The step of using an antibody is included.

At least 95% identical to a nucleotide sequence that encodes a polypeptide that includes an amino acid sequence that is at least 90% identical to a sequence of at least 10 consecutive amino acids in a sequence selected from the group consisting of: Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence: the amino acid sequence of SEQ ID NO: (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.
The complete amino acid sequence of the secretory protein encoded by the human cDNA clone contained in the deposit with the CC accession number.

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

Also preferred is an isolated nucleic acid molecule wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO: Y, where Y is any of those defined in Table 1. Is an integer); and the complete amino acid sequence of the secretory protein encoded by the human cDNA clone identified in the cDNA clone ID in Table 1 and included in the deposit with the ATCC deposit number shown for this cDNA clone in Table 1.

Further preferred is a method of making a recombinant vector comprising the step of inserting any of the above isolated nucleic acid molecules into a vector. The recombinant vector produced by this method is also preferred. Also preferred is a method of making a recombinant host cell, which comprises the step of introducing the vector into a host cell, as well as a recombinant host cell produced by this method.

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

Also preferred are methods of treating an individual in need of increased levels of secreted protein activity. Here, the method comprises administering to such an individual a fixed amount of an isolated polypeptide, polynucleotide, or antibody of the invention effective to increase the level of activity of this protein in the individual. Administering the topical composition.

The applications cited above 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-human primates, and humans are included, but are not limited to. In a particular embodiment, 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 the most preferred embodiment, the host is human.

In a particular embodiment of the present invention, for each “Contig ID” listed in the fourth column of Table 7, preferably the nucleotide sequence referenced in the fifth column of Table 7 and the general formula one or more comprising or consisting of a nucleotide sequence described by ab, where a and b are uniquely determined for the corresponding SEQ ID NO: X referenced in column 3 of Table 7. Polynucleotides are excluded. A more particular embodiment is directed to polynucleotide sequences that exclude 1, 2, 3, 4, or more of the particular polynucleotide sequences referenced in the fifth column of Table 7. This table is in no way meant to include all of the sequences that can be excluded by the general formula, which is merely an illustrative example. All documents available through these registries are hereby incorporated by reference in their entireties.

[0677]

[Table 7] Although the invention has been generally described, the invention will be more readily understood by reference to the following examples, which are provided for purposes of illustration and are not intended to be limiting.

EXAMPLES Example 1: Isolation of Selected cDNA Clones from Deposited Samples Each cDNA clone in the referenced ATCC deposit is contained in a plasmid vector. Table 1 identifies the vector used to construct the cDNA library from which each clone was isolated. In many cases, the vector used to construct the library is a plasmid excised phage vector. The table immediately below correlates the relevant plasmids for each phage vector used in constructing the cDNA library. For example, if a particular clone is identified in Table 1 as being isolated in the vector "Lambda Zap", the corresponding deposited clone is "pBluescript".

[0679]

[Table 8] Vector Lambda Zap (US Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (US Pat. No. 5,128,25).
6 and 5,286,636), Zap Express (US Pat. Nos. 5,128,256 and 5,286,636), pBluescri.
pt (pBS) (Short, JM et al., Nucleic Acids Re).
s. 16: 7583-7600 (1988); Alting-Mees, M .; A
． And Short, J. et al. M. , Nucleic Acids Res. 17
: 9494 (1989)) and pBK (Alting-Mees, MA).
Et al., Strategies 5: 58-61 (1992)) by Stratag.
ene Cloning Systems, Inc. , 11011 N.I. Tor
commercially available from rey Pines Road, La Jolla, CA, 92037. pBS contains the ampicillin resistance gene and pBK contains the neomycin resistance gene. Both are E. E. coli strain XL-1 Blue, which is also available from Stratagene. pB
S arrives in four forms: SK +, SK-, KS + and KS. S and K are polylinker regions (“S” is SacI and “K” is KpnI).
Which is the first site at each end of each of the linkers)
Refers to the orientation of the polylinker relative to the T7 and T3 primer sequences flanking.
"+" Or "-" means the orientation of the f1 origin of replication ("ori") such that single-stranded rescue initiated from f1 ori in one direction produces sense strand DNA, and antisense in the other. Say.

Vectors pSport1, pCMVSport 2.0 and pCMVSpo
rt3.0 from Life Technologies, Inc. , P .; O. Box
6009, Gaithersburg, MD 20897. All Sport vectors contain the ampicillin resistance gene, and E. coli. coli strain D
H10B, which is also available from Life Technologies, can be transformed. (For example, Gruber, CE et al., Focus)
15:59 (1993)). Vector lafmid BA (Ben
to Soares, Columbia University, NY) contains the ampicillin resistance gene, and E. E. coli strain XL-1 Blue. Vector pCR® 2.1 (this is Invitroge
n, 1600 Faraway Avenue, Carlsbad, CA 92
(Available from E. 008) contains an 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 .; Et al, Bi
o / Technology 9: (1991)). Preferably, the polynucleotides of the invention do not include the phage vector sequences identified for the particular clones in Table 1, as well as the corresponding plasmid vector sequences shown above.

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

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

In particular, a particular polynucleotide having 30-40 nucleotides is synthesized according to the reported sequence using an Applied Biosystems DNA synthesizer. The oligonucleotide is, for example, ³² P-γ-ATP,
Labeled with T4 polynucleotide kinase and purified according to conventional methods. (For example, Maniatis et al., Molecular Cloning.
: A Laboratory Manual, Cold Spring Har
bor Press, Cold Spring, NY (1982)). The plasmid mixture is prepared using techniques known to those of skill in the art (eg, those provided by vector suppliers or the relevant publications or patents cited above) in an appropriate host as described above. (For example, XL-1 Blue (Stra
transformation). Transformants are plated on 1.5% agar plates (containing a suitable selection agent such as ampicillin) at a density of about 150 transformants (colony) per plate. These plates were subjected to conventional methods for bacterial colony screening (eg Sambrook et al.,
Molecular Cloning: A Laboratory Manual
1, Second Edition, (1989), Cold Spring Harbor Labo.
rattle press, 1.93-1.104) or other techniques known to those of skill in the art to screen using nylon membranes.

Alternatively, at both ends of SEQ ID NO: X (ie, the 5'N of the clones defined in Table 1).
17-2 from within the region of SEQ ID NO: X surrounded by T and 3′NT)
Two primers of 0 nucleotides are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under conventional conditions, for example in 25 μl of a reaction mixture with 0.5 μg of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl ₂ , 0.01% (w / v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 units of Taq polymerase. . 35 cycle PC
R (denaturation at 94 ° C. for 1 min; annealing at 55 ° C. for 1 min; extension at 72 ° C. for 1 min) is performed using a Perkin-Elmer Cetus automated thermal cycler. The amplification product is analyzed by agarose gel electrophoresis and the DNA band of the expected molecular weight is excised and purified. PCR product, D
Confirm the selected sequence by subcloning and sequencing the NA product.

Several methods are available for identification of the 5'non-coding or 3'non-coding portions of genes that may not be present in the deposited clone. These methods include, but are not limited to: filter probe probing, 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)).

Briefly, specific RNA oligonucleotides are ligated to the 5'end of a population of RNAs that probably contains full-length gene RNA transcripts. PCR of the 5'portion of the desired full-length gene is performed using a primer set containing a primer specific to the linked RNA oligonucleotide and a primer specific to the known sequence of the gene of interest.
Amplify. The amplified product can then be sequenced and used to generate the full length gene.

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

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

Example 2 Isolation of Genomic 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 Tissue distribution of mRNA expression of the polynucleotides of the present invention, particularly Samb.
It is determined using the protocol for Northern blot analysis described by look et al. For example, the cDNA produced by the method described in Example 1.
The probe was applied to the rediprime ^™ DNA labeling system.
Label with P ³² using m (Amersham Life Science) according to the manufacturer's instructions. After labeling, the probe was placed on CHROMA SPIN-1.
Purify using a 00 ^™ column (Clontech Laboratories, Inc.) according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to test various human tissues for mRNA expression.

Multiple Tissue Northern (MTN) blots (Clontech) containing different human tissues (H) or human immune system tissues (IM) were used with ExpressHyb ^™ Hybridization Solution (Clontech), manufacturer's protocol number PT1190. Test with labeled probe according to -1. After hybridization and washing, the blots are mounted and exposed to film overnight at -70 ° C and the film developed according to standard procedures.

Example 4 Polynucleotide Chromosome Mapping A set of oligonucleotide primers is designed according to the sequence at the 5 ′ end of SEQ ID NO: X. This primer preferably spans about 100 nucleotides.
This primer set is then used for polymerase chain reaction under the following set of conditions: 95 ° C for 30 seconds; 56 ° C for 1 minute; 70 ° C for 1 minute. This cycle is repeated 32 times and then once at 70 ° C. for 5 minutes. Somatic cell hybrid panel 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 is performed for about 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 are PCR oligos corresponding to the 5 ′ and 3 ′ ends of the DNA sequences, as outlined in Example 1. Amplification is performed using nucleotide primers to synthesize the insert fragment. The primer used to amplify the cDNA insert should preferably contain restriction sites such as BamHI and XbaI at the 5'end of the primer for cloning the amplification product into an expression vector. For example, BamHI and XbaI are bacterial expression vectors pQE-9 (Qiagen, Inc., Chatsworth,
CA) corresponding to the restriction enzyme site. This plasmid vector
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 maintaining the reading frame initiated in bacterial RBS. The ligation mixture was then treated with the plasmid pR, which expresses the lacI repressor and also confers kanamycin resistance (Kan ^r ).
E. coli, including multiple copies of EP4. E. coli strain M15 / rep4 (Qiagen,
Inc. ). Transformants are identified by their ability to grow on LB plates and ampicillin / kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.

Clones containing the desired construct were cloned into Amp (100 μg / ml) and Kan (100 μg / ml).
Liquid culture in LB medium supplemented with both (25 μg / ml) overnight (O / N)
Proliferate. The O / N culture is used to inoculate a large culture at a ratio of 1: 100 to 1: 250. The cells were placed at an optical density of 600 (OD) between 0.4 and 0.6.
． Grow to ⁶⁰⁰ ). Then IPTG (isopropyl-BD-thiogalactopyranoside) is added to a final concentration of 1 mM. IPTG is lac
Induced by inactivation of the I repressor, clears P / O obstructions and leads to increased gene expression.

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

Briefly, the supernatant was loaded onto a column of 6M Guanidine-HCl, pH8, which column was first washed with 10 volumes of 6M Guanidine-HCl, pH8,
It is then washed with 10 volumes of 6M guanidine-HCl, pH 6, and finally the polypeptide is eluted with 6M guanidine-HCl, pH 5.

The purified protein was then loaded with 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 the Ni-NTA column. Recommended conditions are as follows: Regeneration using a linear gradient of 6M to 1M urea in 500 mM NaCl, 20% glycerol, 20 mM Tris / HCl pH 7.4 with protease inhibitors. Regeneration should take a minimum of 1.5 hours. After playing
The protein is eluted by the addition of 250 mM imidazole. Imidazole in PBS or 50 mM sodium acetate pH 6 buffer and 200 mM
It is removed by a final dialysis step against NaCl. 4 purified proteins
Store at ℃ or frozen at -80 ℃.

In addition to the expression vectors described above, the invention further comprises an expression vector called pHE4a, which contains a phage operator and promoter elements operably linked to the polynucleotide of the invention (ATCC Accession No. 209645).
, Deposited on February 25, 1998). This vector contains: 1) the neomycin phosphotransferase gene as a selectable marker, 2) E. 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 was restricted with NdeI and XbaI, BamHI, XhoI, or Asp718, the restriction products were run on a gel, and the larger fragment (the stuffer fragment should be approximately 310 base pairs). DNA) can be inserted into pHEa by isolating D
The NA insert was labeled with NdeI (5 ′ primer) and XbaI, BamHI, Xh.
PCR with restriction sites for oI or Asp718 (3 'primer)
Primers are used and generated according to the PCR protocol described in Example 1.
The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.

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

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

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

The cells were then transferred to a microfluidizer (
Microfluidics, Corp. Or APV Gaulin, Inc
． ) Is dissolved twice by passing the solution at 4000-6000 psi.
The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000 xg for 15 minutes. The obtained pellet was treated with 0.5 M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.
Wash again using 4.

The resulting washed inclusion bodies were treated with 1.5M guanidine hydrochloride (GuHCl) to
Solubilize for 4 hours. After centrifugation at 7000 xg for 15 minutes, the pellet is discarded, and the polypeptide-containing supernatant is incubated at 4 ° C overnight to allow additional Gu.
Allows HCl extraction.

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

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

Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample was then added to a pre-prepared strong anion (Poros HQ
-50, Perseptive Biosystems exchange resin and weak anion (Poros CM-20, Perseptive Biosystems).
) Load into a set of in-line columns of exchange resin. The column is equilibrated with 40 mM sodium acetate, pH 6.0. Both columns were loaded with 40 mM sodium acetate,
Wash with 200 mM NaCl, pH 6.0. Next, the CM-20 column is replaced with 10
Linear gradient of column volume (0.2 M NaCl, 50 mM sodium acetate, pH
Elution with 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5). Fractions are collected under constant _A280 monitoring of the eluate. Fractions containing the polypeptide (eg, found by 16% SDS-PAGE) are then pooled.

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

Example 7: Cloning and Expression of Polypeptides in Baculovirus Expression System In this example, plasmid shuttle vector pA2 is used to insert a polynucleotide into a baculovirus to express a polypeptide. This expression vector is used for Autographa californica nuclear polyhedrosis virus (
AcMNPV) strong polyhedrin promoter followed by BamHI, Xba
I, and convenient restriction sites such as Asp718. Simian virus 40
The polyadenylation site of ("SV40") is used for efficient polyadenylation. For easy selection of recombinant virus, this plasmid was engineered into E. coli under the control of the weak Drosophila promoter in the same orientation. 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, which produces a viable virus expressing the cloned polynucleotide.

Many other baculovirus vectors (eg pAc373, pVL941)
, And pAcIM1) are signals that the construct is properly positioned for transcription, translation, secretion, etc. (including signal peptide and in-frame AUG, if required), as will be readily appreciated by those of skill in the art. Can be used in place of the above vector as long as Such a vector is, for example, Lucco.
W et al., Virology 170: 31-39 (1989).

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

The amplified fragment was transferred to a commercially available kit (“Geneclean”, BIO
101 Inc. , La Jolla, Ca. ) Is used to isolate from a 1% agarose gel. This fragment is then digested with the appropriate restriction enzymes and again purified on a 1% agarose gel.

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

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

5 μg of plasmid containing this polynucleotide was prepared according to the method of Felgner et al., Pr.
oc. Natl. Acad. Sci. USA 84: 7413-7417 (19).
87) and 1.0 μg of commercially available linearized baculovirus DNA (“BaculoGold ^™ baculov”).
irus 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
chnologies Inc. , Gaithersburg, MD),
Mix in sterile wells of a microtiter plate. Thereafter, 10 μl Lipofectin and 90 μl Grace's medium were added, mixed and allowed to stand at room temperature for 15
Incubate for minutes. The transfection mixture is then added dropwise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated at 27 ° C for 5 hours. The transfection solution is then removed from the plate and 1 ml Grace's insect medium supplemented with 10% fetal bovine serum is added. The culture is then continued for 4 days at 27 ° C.

Supernatants are collected after 4 days and plaque assay performed as described by Summers and Smith (supra). "Blue Gal" (Life
Technologies Inc. , Gaithersburg) was used to allow easy identification and isolation of gal-expressing clones (resulting in blue-stained plaques) (details of this type of "plaque assay"). The description is also provided by Life Technologies Inc.,
Gaithersburg, pages 9-10, which can be found in the user guide for insect cell culture and baculovirology). After appropriate incubation, blue stained plaques are picked up with a micropipette tip (eg Eppendorf). The agar containing the recombinant virus is then added to 2
Resuspend in a microcentrifuge tube containing 00 μl Grace's medium and use the suspension containing recombinant baculovirus to infect Sf9 cells seeded in 35 mm dishes. After 4 days, the supernatants of these culture dishes are collected and then stored at 4 ° C.

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

Micro-sequencing of the amino-terminal amino acid sequence of purified proteins can be used to determine the amino-terminal sequence of the produced protein.

Example 8 Expression of Polypeptides in Mammalian Cells The polypeptides of the present invention can be expressed in mammalian cells. A typical mammalian expression vector contains promoter elements that mediate the initiation of transcription of mRNA, protein coding sequences, and signals necessary for termination of transcription and polyadenylation of the transcript. Further elements are enhancers, Kozak (Koz
ak) sequences and intervening sequences flanking the donor and acceptor sites for RNA splicing. Very efficient transcription is achieved by the SV40-derived early and late promoters, retroviruses (eg RSV, HTLVI, H
IVI) -derived long terminal repeat (LTR), and cytomegalovirus (CMV)
Is achieved using the early promoter of. However, intracellular elements (eg,
The human actin promoter) can also be used.

Expression vectors suitable for use in practicing the present invention are, for example, pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVc.
at (ATCC 37152), pSV2dhfr (ATCC 37146),
Includes vectors such as pBC12MI (ATCC 67109), pCMVSport2.0, as well as pCMVSport3.0. Mammalian host cells that can be used include human Hela cells, 293 cells, H9 cells and Jurka.
t cells, mouse NIH3T3 cells and C127 cells, Cos 1 cells, Co
s 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 a chromosome,
It can be expressed in stable cell lines. Co-transfection with selectable markers such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of transfected cells.

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

Expression vectors pC4 (ATCC Accession No. 209646) and pC6 (AT) which are derivatives of plasmid pSV2-dhfr (ATCC Accession No. 37146).
CC Accession No. 209647) is for Rous sarcoma virus (Cullen et al., Mol.
electrical and Cellular Biology, 438-447 (
March 1985)), a strong promoter (LTR), and a fragment of the CMV-enhancer (Boshart et al., Cell 41: 521-530 (1985)). For example, multiple cloning sites with BamHI, XbaI, and Asp718 restriction enzyme cleavage sites facilitate cloning of the gene of interest. This vector also contains a 3'intron, polyadenylation and termination signals of the rat preproinsulin gene, and S
Contains the mouse DHFR gene under the control of the V40 early promoter.

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

Polynucleotides of the invention are amplified according to the protocol outlined in Example 1. If a naturally occurring signal sequence is used for the production of secreted protein, the vector need not include a second signal peptide. Alternatively,
If the naturally-occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence (see, eg, WO96 / 34891).

The amplified fragment was transferred to a commercially available kit (“Geneclean”, BIO 10
1 Inc. , La Jolla, Ca. ) Is used to isolate from a 1% agarose gel. This fragment is then digested with the appropriate restriction enzymes and again purified 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 using, for example, restriction enzyme analysis.

Chinese hamster ovary cells lacking the active DHFR gene are used for transfection. 5 μg of the expression plasmid pC6 a pC4 was transformed with 0.5 μg of plasmid pC6a pC4 using lipofectin (Felgner et al.
Co-transfect with SVneo. Plasmid pSV2-neo contains the dominant selectable marker, the Tn5-derived neo gene encoding an enzyme that confers resistance to a group of antibiotics, including G418. Cells are seeded in α-minus MEM supplemented with 1 mg / ml G418. After 2 days, cells were trypsinized and treated with 10, 25, or 50 ng / ml methotrexate and 1 m.
Seed in hybridoma cloning plates (Greiner, Germany) in α-MEM supplemented with g / ml G418. After about 10-14 days, single clones were trypsinized and then using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).
Seed in 6-well Petri dishes or 10 ml flasks. Clones growing at the highest concentration of methotrexate were then selected for 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 clones are obtained that grow at a concentration of 100-200 μM. Expression of the desired gene product is analyzed, for example, by SDS-PAGE and Western blot, or by reverse phase HPLC analysis.

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

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

For example, if pC4 (accession number 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3'BamHI site should be destroyed. Then, the vector containing the human Fc portion is transformed into B
The polynucleotide of the invention, which was again restricted with amHI, linearized the vector and isolated by the PCR protocol described in Example 1,
It is linked to the amHI site. Note that the polynucleotide is cloned without a stop codon, otherwise no fusion protein will be produced.

PC4 does not require a second signal peptide if a naturally occurring signal sequence is used to produce the secreted protein. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence (see, eg, WO 96/34891). Human IgG Fc region: GGGATCCGGAGCCCAAATCTTCTGACAAAAACTCACA
CATGCCCACCGTGCCCAGCACCTGAATTCGAGGGGTG
CACCGTCAGTCTTCCCCTTCCCCCCCAAAAACCCAAGG
ACACCCTCATGATCTCCCGGACTCCTGAGGTCACAT
GCGTGGGTGGGGACGTAAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACCGTGGACGGCGTGGAGGGTGC
ATAATGCCAAGACAAAGCCGCGGGAGGAGGCAGTACA
ACAGCACGTACCCGTGTGGTCAGCGTCTCACCACCGTCC
TGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGT
GCAAGGTCTCCAACAAAGCCCTCCCAACCCCCCATCG
AGAAAACCCATCTCCAAAGCCAAAGGGCAGCCCCCGAG
AACCACAGGTGTACACCCTGCCCCCCATCCCGGGATG
AGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGG
TCAAAGGCTTCTATCCAAAGCGACCATCGCCGTGGAGGT
GGGAGAGCAATGGGGCAGCCGGAGAACAACTACAAGA
CCACGCCTCCCGTGCTGGACTCCGACGCGCTCCTTCT
TCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGT
GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC
ATGAGGCTCTGCACACACCACTACACCGCAGAAGAGCC
TCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGC
GACTCTAGAGGAT (SEQ ID NO: 1) (Example 10: Production of antibody from polypeptide) The antibody of the present invention can be prepared by various methods. (Current Pr
otocols, 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 secreted protein is prepared and purified to be substantially free of natural contaminants. Such preparations are then introduced into animals to produce greater specific activity of polyclonal antisera.

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

The splenocytes of such mice are extracted and fused with the appropriate myeloma cell line. Any suitable myeloma cell line may be used in accordance with the present invention; however, AT
It is preferred to use the parental myeloma cell line (SP2O) available from CC. After fusion, the resulting hybridoma cells were selectively maintained in HAT medium and then Wands et al. (Gastroenterology 80: 225-232 (
Cloning by limiting dilution as described by 1981)). The hybridoma cells obtained through such selection are then assayed to identify clones secreting antibodies capable of binding the polypeptide.

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

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 generated by proteolytic cleavage using an enzyme such as papain (to produce Fab fragments) or pepsin (to produce F (ab ') 2 fragments). To be done. Alternatively, secreted protein-binding fragments may be produced by the application of recombinant DNA technology or synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use "humanized" chimeric monoclonal antibodies. Such antibodies can be produced by using genetic constructs derived from hybridoma cells which produce the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. (For a review, see Morrison, Science 229: 1202.
(1985); Oi et al., BioTechniques 4: 214 (1986).
Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neube.
rger et al., WO 8601533; Robinson et al., WO 870267.
1; Boulianne et al., Nature 312: 643 (1984); Ne.
Uberger et al., Nature 314: 268 (1985)).
.

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

First, poly-D-lysine (644 587 Boehringer-Man).
nheim) stock solution (1 mg / ml in PBS) in PBS (17-516F Biowhittaker 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. Make sure to distribute the solution over each well (Note: a 12 channel pipettor can be used with tips on every other channel).
Aspirate the poly-D-lysine solution and rinse with 1 ml PBS (phosphate buffered saline). PBS should remain in the wells just prior to plating the cells, and the plates can be pre-coated with polylysine by 2 weeks.

293T cells (passing P + 20 and not bearing cells) were treated with 2 × 10 ⁵ cells / well in 0.5 ml DMEM (Dulbecco's Modified Eagle Medium) (4.
5 G / L glucose and L-glutamine (12-604F Biowhit
10% heat-inactivated FBS (14-503F Biowhi)
ttaker) / 1 × Penstrep (17-602E Biohitter)
plate). Allow cells to grow overnight.

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

[0743] Preferably, transfection is carried out by forming a tag team (
tag-teaming). By forming a tag team,
The time effort is halved, and the cells do not spend too much time on PBS. First, Person A aspirates the medium from the four 24-well plates of cells, and then Person B rinses each well with 0.5-1 ml PBS. Mr. A then aspirates the PBS rinse and Mr. B uses 200 μl of DNA using a 12-channel pipettor with tips on every other channel.
/ 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.

While incubating the cells, either 1% BSA in DMEM with 1 × penstrep or CHO-5 medium (116.6 mg / L).
CaCl ₂ (anhydrous); 0.00130 mg / L CuSO ₄ -5H ₂ O; 0
． 050mg / L of _{Fe (NO 3) 3 -9H 2} O; 0.417mg / L FeS of
_{O 4 -7H 2 O; 311.80mg /} L of KCl; 28.64mg / L MgC of
l _2; 48.84mg / L MgSO ₄ for; 6995.50mg / L of NaCl;
NaHCO of 2400.0mg / L _3; 62.50mg / L of _NaH 2 PO ₄ -
H ₂ O; 71.02 mg / L Na ₂ HPO ₄ ; 0.4320 mg / L ZnS
O ₄ -7H ₂ O; 0.002 mg / L arachidonic acid; 1.022 mg / L cholesterol; 0.070 mg / L DL-α-tocopherol acetate; 0.05
20 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 (palmitic acid); 0.010 mg / L palmitic acid; 100 mg / L Pluronic F-68; 010 mg
/ L stearic acid; 2.20 mg / L Tween 80; 4551 mg / L D-glucose; 130.85 mg / ml L-alanine; 147.50 mg /
ml of L- arginine -HCl; 7.50mg / 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. 29 mg / ml L-cystine-2HCl; 7
． 35 mg / ml L-glutamic acid; 365.0 mg / ml L-glutamine; 18.75 mg / ml glycine; 52.48 mg / ml L-histidine-
_{HCl-H 2 O; 106.97mg /} ml of L- isoleucine; 111.45M
32.g / ml L-leucine; 163.75 mg / ml L-lysine HCl;
34 mg / ml L-methionine; 68.48 mg / ml L-phenylalanine; 40.0 mg / ml L-proline; 26.25 mg / ml L-serine;
101.05 mg / ml L-threonine; 19.22 mg / ml L-tryptophan; 91.79 mg / ml L-tyrosine-2N.
_{a-2H 2 O; 99.65mg /} ml of L- valine; 0.0035 mg / L biotin; 3.24mg / L D-Ca pantothenate; 11.78mg / L choline chloride; 4.65mg / L Folic acid; 15.60 mg / L i-inositol; 3
． 02 mg / L niacinamide; 3.00 mg / L pyridoxal HCl
0.031 mg / L pyridoxine HCl; 0.319 mg / L riboflavin; 3.17 mg / L thiamine HCl; 0.365 mg / L thymidine; and 0.680 mg / L vitamin B ₁₂ ; 25 mM HEPES buffer. Agent; 2.3
9 mg / L Na hypoxanthine; 0.105 mg / L lipoic acid; 0.081 m
g / L sodium putrescine-2HCl; 55.0 mg / L sodium pyruvate; 0.0067 mg / L sodium selenite; 20 μM ethanolamine; 0.122 mg / L ferric citrate; 41.70 mg Methyl-B-cyclodextrin complexed with / L linoleic acid; Methyl-B-cyclodextrin complexed with 33.33 mg / L oleic acid; and 10 mg / L
Retinal complex of Methyl-B-cyclodextrin). (For a 10% BSA stock solution, 100 g of BSA (81-068-3 Bayer) was dissolved in 1 L DMEM). Media is filtered and 50 μl collected in 15 ml polystyrene conical for endotoxin assay.

The transfection reaction is terminated at the end of the incubation time, preferably in tag teams. Person A aspirates off the transfection medium, while person 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
Aliquot into 1 ml deep well plate and 2 ml of remaining supernatant
Aliquot into deep wells of. The supernatant from each well was then added to Examples 13-2.
0 may be used in the assay described in 0.

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

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

GAS and ISRE elements are recognized by signal transducers and activators of transcription, a class of transcription factors called “STATs”. There are 6 members in the STAT family. S
tat1 and Stat3, as well as Stat2 (as the response to IFNα is widespread) are present in many cell types. Stat4 is more restricted and is not present in many cell types, but is found in T helper class I cells after treatment with IL-12. Stat5, originally called breast growth factor, is found in 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.

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 cytokine receptor family that is capable of activating sac can be divided into two groups:
(A) Class 1 includes IL-2, IL-3, IL-4, IL-6, IL-7, IL
-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CS
F, GM-CSF, LIF, CNTF, and receptors for thrombopoietin; and (b) class 2 includes IFN-a, IFN-g, and IL-.
Including 10. Class 1 receptors contain a conserved cysteine motif (a set of 4 conserved cysteines and 1 tryptophan), and WSXWS.
Shares the motif (membrane adjacent region encoding Trp-Ser-Xxx-Trp-Ser (SEQ ID NO: 2)).

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

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

[0754]

[Table 9] To construct synthetic GAS containing promoter elements used in the biological assays described in Examples 13-14, a PCR-based strategy is used to generate the GAS-SV40 promoter sequence. 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 flanks the XhoI site. The sequence of the 5'primer is: 5 ': GCGCCTCGAGATTTTCCCCGAAATCTAGATTTTCC
CCGAAATGATTTTCCCCGAAATGATTTTCCCCGAAATA
TCTGCCATCTCAATTAG: 3 '(SEQ ID NO: 3).

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

PCR amplification is performed with 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 with forward and reverse primers confirms that the insert contains the following sequence: 5 ': CTCGAG ATTTCCCCGAAATCTAGATTTCCCCGA.
AATGATTTTCCCCGAAATGATTTCCCGAAATATCTG
CCATCTCAATTTAGTCAGCAACCATATAGTCCCGCCCCT
AACTCCGCCCCATCCCGCCCCTAACTCCGCCCAGTTC
CGCCCATTCTCCGCCCCATGGCTGAACTAATTTTTTTT
TATTTATGCAGAGGCCGAGGCCGCCTCGCGCTCTGA
GCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGG
CCTAGGCTTTTGCAAA AAGCTT: 3 '(SEQ ID NO: 5) When this GAS promoter element is coupled to the SV40 promoter,
The GAS: SEAP2 reporter construct is then engineered. Here, the reporter molecule is secretory alkaline phosphatase or "SEAP". However, clearly any reporter molecule can substitute for SEAP in either this or other examples. Well-known reporter molecules that can be used in place of SEAP include chloramphenicol acetyl transferase (C
AT), luciferase, alkaline phosphatase, β-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.

Synthetic GAS-SV40 promoter elements identified by the above sequence were added to HindIII and XhoI to generate 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.

Therefore, in order to generate a stable mammalian cell line expressing the 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 in the multiple cloning site were used to construct a backbone vector containing the neomycin resistance gene (eg pGFP-1 (C
longtech)). Once the vector is 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 substituting GAS with a different promoter sequence. For example, the construction of a reporter molecule containing 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, the 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), HUVEC (endothelial cells), Reh (B cells), Saos-2 (osteoblasts), HUVAC (aortic cells), or cardiomyocytes) can be used as reporters. It can be used to test the activity of the construct.

Example 13 High-Throughput Screening Assay for Activity of T Cells The following protocol was used to identify agents and supernatants containing the polypeptides of the invention expanded T cells and T cell activity is assessed by determining whether to differentiate / or differentiate. GA of T cell activity produced in Example 12
Assess with the S / SEAP / Neo construct. Thus, factors that increase SEAP activity show the ability to activate the Jaks-STATS signaling pathway.
The T cells used in this assay are Jurkat T cells (ATCC Accession No. T
IB-152) but Molt-3 cells (ATCC Accession No. CRL-155).
2) and Molt-4 cells (ATCC Accession No. CRL-1582) cells may also be used.

Jurkat T cells are lymphoblastic CD4 + Th1 helper cells. Approximately 2 million Jurkat cells were DMRed to generate stable cell lines.
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 of geneticin are selected. Growing resistant colonies, then
Test for their response to increasing concentrations of interferon-γ. The dose response of selected clones is shown.

In detail, the following protocol yields sufficient cells for 75 wells containing 200 μl of cells. Therefore, in order to produce sufficient cells for multiple 96-well plates, this is either scaled up or performed in multiples. Jurkat cells in 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 at room temperature for 15-45 minutes.

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 /
Resuspend in OPTI-MEM to make ml. Then, OPTI-MEM
1 ml of 1 × 10 ⁷ cells in is added to a T25 flask and incubated at 37 ° C. for 6 hours. After incubation 10 ml RPMI + 15%
Serum is added.

Jurkat: GAS-SEAP stable reporter strain was added to RPMI + 10% serum,
Maintain in 1 mg / ml Geneticin, and 1% Pen-Strep.
These cells are treated with a supernatant containing a polypeptide of the invention and / or are induced with a 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 500,00 ml / ml
It should be resuspended in fresh RPMI + 10% serum to a density of 0 cells. The exact number of cells required depends on the number of supernatants screened.
Approximately 10 million cells are needed per 96-well plate (100 million cells per 10 plates).

Transfer the cells to a triangular reservoir boat to dispense the 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 0,000 cells).

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

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

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

The above 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 proliferates and / or differentiates myeloid cells. Evaluate myeloid activity. The activity of myeloid cells is assessed using the GAS / SEAP / Neo construct produced in Example 12. Therefore, a factor that increases SEAP activity is Jaks-S.
Shows the ability to activate the TATS signaling pathway. The myeloid cells used in this assay can be U937 (which is a pre-monocyte cell line), TF-1, HL60, or KG1.

The DEAE-Dextran method (for transient transfection of U937 cells with the GAS / SEAP / Neo construct produced in Example 12).
Kharbanda et al., 1994, Cell Growth & Differ.
Orientation, 5: 259-265). First, 2 × 10e ⁷ U937 cells are harvested 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 GAS-SE.
AP2 plasmid DNA, 140 mM NaCl, 5 mM KCl, 375 μM
_{Na 2 HPO 4 · 7H 2 O} , 1mM MgCl 2, and 675MyuM CaC
the cells are suspended in 20mM Tris-HCl (pH 7.4) in buffer 1ml containing l _2. Incubate at 37 ° C for 45 minutes.

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

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

These cells are tested by harvesting 1 × 10 ⁸ cells, which is sufficient for 10 96-well plate assays, and washing with PBS. Suspend the cells in the above 200 ml of growth medium at a final density of 5 × 10 ⁵ cells / ml. Plate 200 μl cells per well (ie 1 × 10 ⁵ cells / well) in a 96-well plate.

Add 50 μl of the supernatant prepared according to the protocol described in Example 11. Incubate at 37 ° C for 48-72 hours. 1 as a positive control
00 units / ml of interferon gamma can be used, which is known to activate U937 cells. Typically,> 30-fold induction is observed in positive control wells. The supernatant is SE according to the protocol described in Example 17.
AP assay.

Example 15: High-Throughput Screening Assay to Identify Neuronal Activity When a cell undergoes differentiation and proliferation, a panel of genes is 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.

In detail, the following protocol is used to assess neuronal activity in the PC12 cell line. PC12 cells (rat pheochromocytoma
ocytoma cells) proliferate and / or differentiate by activation with many mitogens such as TPA (tetradecanoylphorbol acetate), NGF (nerve growth factor), and EGF (epithelial growth factor). Is known. EGR1 gene expression is activated during this treatment. Therefore, PC12 cell activation can be assessed by stably transfecting PC12 cells with a construct containing an EGR promoter linked to a SEAP reporter.

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

The GAS: SEAP / Neo vector generated in Example 12 can then be used to insert the EGR1 amplification product into this vector. Restriction enzyme XhoI / H
Linearize the GAS: SEAP / Neo vector using indIII
Remove the S / SV40 stuffer. EGR1 amplification products are restricted with these same enzymes. Connect the vector and the EGR1 promoter.

To prepare 96-well plates for cell culture, coating solution (
Collagen type I (Upstate Biotech Inc. Catalog No. 08
-115) diluted 1:30 with 30% ethanol (sterilized filtration) 10 cm each
Add 2 ml per plate, or 50 ml per well in a 96-well plate and allow to air dry for 2 hours.

PC12 cells were plated on a 10 cm tissue culture dish pre-coated with 10% horse serum (JRH BIOSCIENCES, catalog # 124) supplemented with penicillin (100 units / ml) and streptomycin (100 μg / ml).
49-78P), RPMI-164 containing 5% heat-inactivated fetal bovine serum (FBS)
Routinely grow in 0 medium (Bio Whittaker). Divide from 1 to 4 every 3-4 days. Peel off the plate by scraping the cells,
Then pipet up and down more than 15 times to resuspend.

The EGR / SEAP / Neo construct was prepared 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 routine growth, but every 1-2 months cells should be regrown in 300 μg / ml G418 for 2 passages.

To assay neuronal activity, 10 cm plates with approximately 70-80% confluent cells are screened by removing old medium. Wash once with PBS (phosphate buffer solution). The cells were then treated with low serum medium (RPMI containing 1% horse serum and 0.5% FBS with antibiotics).
-1640) starve the cells overnight.

The next morning, remove the medium and wash the cells with PBS. The cells are scraped from the plate and the cells are suspended in 2 ml of low serum medium. The number of cells is counted and additional medium with low serum is added to reach a final cell density of 5 × 10 ⁵ cells / ml.

200 μl of cell suspension is added to each well of a 96-well plate (1 × 1
0 ⁵ cells / well). 50 μl of the supernatant produced according to Example 11
Add at 37 ° C for 48-72 hours. As a positive control, P via EGR
Growth factors known to activate C12 cells can be used, such as 50 ng / μl nerve growth factor (NGF). Typically,> 50-fold SEAP induction is found in positive control wells. The supernatant is treated with SE according to Example 17.
AP assay.

Example 16: High-Throughput Screening Assay for T Cell Activity NF-κB (Nuclear Factor κB) was found in a wide variety of agents (inflammatory cytokines IL-1 and TNF, CD30 and CD40, (Including lymphotoxin-α and lymphotoxin-β), transcription factors that are activated by exposure to LPS or thrombin, and by expression of specific viral gene products. As a transcription factor, NF-κB is the expression of genes involved in the activation of immune cells, regulation of apoptosis (NF-κB appears to protect cells from apoptosis), development of B cells and T cells, antiviral It regulates responses and antimicrobial responses, as well as multiple stress responses.

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

Due to its central role and ability to respond to a range of stimuli, NF-
A reporter construct utilizing the κB promoter element is used to screen the supernatant produced in Example 11. Activators or inhibitors of NF-κB are useful in treating disease. For example, NF-κB inhibitors may be used to treat diseases associated with acute or chronic NF-κB activation, such as rheumatoid arthritis.

To construct a vector containing the NF-κB promoter element, PCR
Use a strategy based on. The upstream primer has an NF-κB binding site (
GGGGACTTTCCC) (SEQ ID NO: 8), containing an 18 bp sequence complementary to the 5'end of the SV40 early promoter sequence and flanked by XhoI sites:
GGGGACTTTCCGGGAACTTTCCATCCTGGCCATCTCAA
TTAG: 3 '(SEQ ID NO: 9).

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

PCR amplification is carried out 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
A sequence with 3 primers confirms that the insert contains the following sequences: 5 ': CTCGAGGGGACTCTCCCGGGGCACTTTCCGGGGGA
CTTTCCGGGACTTTCCATCTGCCATCTCAATTAGTC
AGCAACCCATAGTCCCGCCCCTAACTCCGCCCCATCCC
GCCCCTAACTCCGCCCAGTTTCCGCCCATTCTCCGCC
CCATGGCTGAACTAATTTTTTTTATTTTATGCAGAGGC
CGAGGCCGCCTCGGCCTCTGAGCTTTCCAGAAGTA
GTGAGGAGGCTTTTTTTGAGAGGCCTAGGGCTTTTGCAA
AAAGCTT: 3 '(SEQ ID NO: 10).

XhoI and HindIII are then used to replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) with this NF-κB / SV40 fragment. However, this vector does not contain the neomycin resistance gene and is therefore not preferred for mammalian expression systems.

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

Once the NF-κB / SV40 / SEAP / Neo vector was prepared, stable Jurkat T cells were 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 wells with 5-10 fold activation observed).

Example 17: Assay for SEAP activity SE as a reporter molecule for the assays described in Examples 13-16.
AP activity was measured by Tropix Phospho-li according to the following general procedure.
Assay using the ght Kit (catalog number BP-400). Troop
The ix Phospho-light Kit is a dilution buffer used below,
An assay buffer and a reaction buffer are provided.

Fill the dispenser with 2.5 × Dilution Buffer and dispense 15 μl of 2.5 × Dilution Buffer into an Optiplate containing 35 μl of supernatant. Seal the plate with a plastic sealer and incubate at 65 ° C for 30 minutes. Keep Optiplates apart to avoid uneven heating.

Cool the sample to room temperature for 15 minutes. Empty the dispenser and fill with assay buffer. Add 50 μl assay buffer and incubate 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. The intensity of the chemiluminescent signal is time-dependent, and it takes about 10 minutes to read 5 plates in the luminometer, so 5 plates are processed each time and the second set after 10 minutes. Should start.

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

[0801]

[Table 10] Example 18: High Throughput Screening Assay Identifies 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, sodium and pH. , And changing the membrane potential is known. These changes can be measured in assays that identify the supernatant that binds to the receptor on a particular cell. The following protocol describes an assay for calcium, but this protocol is easily modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule detectable by a fluorescent probe. Can be done.

The following assay measures changes in fluorescent molecules (Molecular Probes) that bind small molecules using a fluorometric imaging plate reader (“FLIPR”). Apparently, any fluorescent molecule that detects small molecules can be used as a calcium fluorescent molecule, fluo-4 (Molecular Probes), as used herein.
, Inc. ; Catalog No. F-14202).

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

A stock solution of 1 mg / ml fluo-4 was added to 10% pluronic acid (pluro acid).
nic acid) Made of DMSO. To load the cells with fluo-4,
12 μg / ml fluo-4 (50 μl) is added to each well. The plate is incubated for 60 minutes at 37 ° C. in a CO ₂ incubator. The plate is washed 4 times with HBSS in the Biotek washer, leaving 100 μl of buffer.

For non-adherent cells, the cells are spun down from the culture medium. 50 cells
resuspended in 2 to 5 × 10 ⁶ cells / ml with HBSS in ml conical tube. F containing 10% DMSO solution of pluronic acid per 1 ml of cell suspension
Add 4 μl of luo-4 (1 mg / ml). The tube is then placed in a 37 ° C water bath for 30-60 minutes. The cells were washed twice with HBSS and 1 × 10 ⁶ cells / ml
Resuspend in 100 μl / well and dispense into microplates. Centrifuge the plate at 1000 rpm for 5 minutes. Then plate the Denley
After washing once with 200 μl in CellWash, the final volume is brought to 100 μl by an 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 the change in fluorescence is detected.

[0807]   In order to measure the fluorescence of intracellular calcium, FLIPR was set to the following parameters.
Set: (1) System gain is 300-800 mW; (2)
Exposure time is 0.4 seconds; (3) camera F / stop is F / 2;
(4) Excitation is 488 nm; (5) Fluorescence is 530 nm; and (6)
Sample addition is 50 μl. The increase in fluorescence at 530 nm is due to intracellular Ca ⁺⁺ Figure 7 shows extracellular signaling events that result in increasing concentrations.

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

Activation of RPTK by a ligand involves ligand-mediated receptor dimerization, which results in transphosphorylation of receptor subunits and activation of cytoplasmic tyrosine kinases. As cytoplasmic tyrosine kinase, src family (
Examples include receptor-related tyrosine kinases of src, yes, lck, lyn, fyn), and non-receptor bound and cell substrate protein tyrosine kinases such as the Jak family. These members include cytokine superfamily (eg, interleukins, interferons, GM-
Mediates signal transduction triggered by receptors for CSF and leptin).

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

Target cells (eg, primary keratinocytes) were purchased from Nalge Nunc (Naperville, IL) at a density of approximately 25,000 cells / well 96.
Seed wells Loprodyne Silent Screen Plate. The plates are sterilized by rinsing twice with 100% ethanol for 30 minutes, rinsed with water and then dried overnight. Several plates were loaded with 100 ml of cell culture grade type I collagen (50 mg / ml), gelatin (2%) or polylysine (
50 mg / ml) (all of these are Sigma Chemicals (St. L.
ouis, MO) or at Becton Dickinso
n (Bedford, MA) 10% Matrigel or calf serum for 2 hours, rinsed with PBS and stored at 4 ° C. Seed 5,000 cells / well containing growth medium and manufacturer Alamar Biosc
iences, Inc. (Sacramento, CA),
By indirect quantification of cell number after 48 hours using lamarBlue,
Cell proliferation is assayed on these plates. Becton Dickins
On (Bedford, MA) Falcon plate cover # 3071 is used to cover the Loprodyne Silent Screen Plate. F
Alcon Microtest III cell culture plates may also be used in some proliferation experiments.

To prepare the extracts, A431 cells are seeded (20,000 / 200 ml / well) on nylon membranes of Loprodyne plates and cultured overnight in complete medium. The cells are incubated in serum-free basal medium for 24 hours to make them rest. 5-2 (50 ng of EGF (60 ng / ml) or the supernatant produced 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, 0.15M NaCl, 1% Triton X-100, 0.
_{1% SDS, 2mM Na 3 VO} 4, 2mM Na 4 P 2 O 7 and Boehe
A mixture of protease inhibitors (# 1836170) from Ringer Mannheim (Indianapolis, Ind.)) is added to each well and the plate shaken on a rotary shaker at 4 ° C. for 5 minutes. Next, the plate is placed on a vacuum transfer manifold.
ifold) and filter the extract using house vacuum on a 0.45 mm membrane at the bottom of each well. 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 a clarified extract by centrifugation, solubilize with detergent for 5 minutes, then remove the contents of each well and centrifuge at 16,000 xg for 15 minutes at 4 ° C.

The filtered extract is tested for levels of tyrosine kinase activity. Although many methods of 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 (cell division kinase cd
and amino acids 6-20 of c2-p34) 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.

The tyrosine kinase reaction is set up by adding the following components in order. First, after adding 10 μl of 5 μM biotinylated peptide, 10 μl of ATP / Mg ²⁺ (5 mM ATP / 50 mM MgCl ₂ ) and 5 × assay buffer (4
0 mM imidazole hydrochloride, pH 7.3, 40 mM β-glycerophosphate, 1 m
M EGTA, 100 mM MgCl ₂ , 5 mM MnCl ₂ , 0.5 mg / m
l BSA) 10 μl, sodium vanadate (1 mM) 5 μl, and finally water 5 μl
Add l. The components are mixed gently and the reaction mixture is preincubated for 2 minutes at 30 ° C. The reaction is started by adding 10 μl of control enzyme or filtered supernatant.

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

Tyrosine kinase activity is measured by transferring a 50 μl aliquot of the reaction mixture to a microtiter plate (MTP) module and incubating at 37 ° C. for 20 minutes. As a result, streptavidin (streptava)
din) Coat 96-well plate with biotinylated peptide.
Wash MTP module 4 times with 300 μl / well PBS. Next, anti-phosphotyrosine conjugated to horseradish peroxidase (phospotyro).
Sine) antibody (anti-P-Tyr-POD (0.5 u / ml)) (75 μl) is added to each well and then incubated at 37 ° C. for 1 hour. Wash wells as above.

Next, a peroxidase substrate solution (Boehringer Mannheim)
) Add 100 μl and incubate at room temperature for at least 5 minutes (up to 30 minutes). The absorbance of the sample at 405 nm is measured using an ELISA reader. The level of bound peroxidase activity was quantified using an ELISA reader, which reflects the level of tyrosine kinase activity.

Example 20: High-Throughput Screening Assay to Identify Phosphorylation Activity Primary cells as potential alternatives and / or complements to 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, p38
Phosphorylation of other molecules such as MAP, Map Kinase Kinase (MEK), MEK Kinase, Src, Muscle Specific Kinase (MuSK), IRAK, Tec and Janus, as well as any other phosphoserine, phosphotyrosine or phosphothreonine molecule. , Erk-1 or E with these molecules in the following assay
It can be detected by replacing rk-2.

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

[0821] A431 cells are seeded at 20,000 / well in 96-well Loprodyne filter plates and cultured overnight in growth medium. Next, the cells are treated with basal medium (D
EGF (6 ng / well) or Example 1 after starvation in MEM) for 48 hours.
Treat with 50 μl of the supernatant obtained in 1 for 5 to 20 minutes. The cells are then solubilized and the extract filtered and placed directly into the assay plate.

After a 1 hour incubation at RT with the extract, the wells are rinsed again. Commercially available MAP kinase preparation (10 ng / well) as a positive control
In place of the A431 extract. The plates are then loaded with Erk-1 and E.
Treatment with a commercially available polyclonal (rabbit) antibody (1 μg / ml) that specifically recognizes a phosphorylated epitope of rk-2 kinase (1 hour at RT). The antibody is biotinylated by standard procedures. The conjugated polyclonal antibody was then added to Wallac D with europium streptavidin and europium fluorescence enhancer.
Quantification by continuous incubation in the ELFIA instrument (time-resolved fluorescence). An increase in fluorescent signal above background indicates phosphorylation.

Example 21: Method for Determining Changes in Genes Corresponding to Polynucleotides RNA isolated from whole families or individual patients presenting with the phenotype of interest (eg, disease) is isolated. CDNA is then generated from these RNA samples using protocols known in the art (see Sambrook).
This cDNA is then used as a template for PCR with primers surrounding the region of interest in SEQ ID NO: X. The suggested PCR conditions are Si
transky, D.I. 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 SequiTherm Polymerase (Epi).
sequence technologies) and sequence at the 5'end with a primer labeled 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. The PCR product with the suspected mutation is then cloned and sequenced to confirm the direct sequencing results.

[0825] The PCR product was analyzed by the method of Holton et al., Nucleic Acids Research.
ch, 19: 1156 (1991) and cloned into a T-tail vector and cloned into T7 polymerase (United States Biochemica).
Sequencing with l). Affected individuals are identified by mutations that are not present in unaffected individuals.

Genomic rearrangements are also observed as a way to determine changes in the gene corresponding to a polynucleotide. Genomic clones isolated according to Example 2 were transformed into digoxigenin deoxy-uridine 5'-triphosphate (Boehringer Man).
Heim) and nick translation, and Johnson et al., M.
methods Cell Biol. 35: 73-99 (1991) and perform FISH. For specific hybridization to the corresponding genomic locus, a large excess of human cot-1 DNA is used to perform hybridization with the labeled probe.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C and R bands. Aligned images for accurate mapping can be generated by triple band filter set (Chroma Tec).
hology, Brattleboro, VT) and cooled charge-coupled device camera (
Photometrics, Tucson, AZ) and tunable excitation wavelength filters (Johnson et al., Genet. Anal. Tech. Appl., 8: 7).
5 (1991)) in combination. ISee Graphical
Program System (Invision Corporation)
on, Durham, NC) using image collection, analysis and chromosomal fractionation (f
ragmental) Length measurement is performed. Chromosomal changes in the genomic region to which the probe hybridizes are identified as insertions, deletions and translocations. These changes are used as diagnostic markers for related diseases.

Example 22: Method for Detecting Abnormal Levels of Polypeptides in Biological Samples Polypeptides of the invention can be detected in biological samples, and elevated or decreased levels of this polypeptide can be detected. If detected, this polypeptide is a marker of a particular phenotype. It is understood that there are numerous detection methods, and therefore one of skill in the art can modify the following assays to suit their particular needs.

For example, an antibody sandwich ELISA is used to detect the polypeptide in a sample, preferably a biological sample. The wells of a microtiter plate are coated with a final concentration of 0.2-10 μg / ml of specific antibody. This antibody is either monoclonal or polyclonal and is described in Example 1.
0 produced by the method described in 0. The well is blocked so that non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated with a sample containing this polypeptide for more than 2 hours at RT. Preferably, serial dilutions of the sample should be used to confirm the results. The plate is then washed 3 times with deionized or distilled water to remove unbound polypeptide.

Next, 50 μl of the specific antibody-alkaline phosphatase conjugate was added to 25-400 n.
Add at a concentration of g and incubate at room temperature for 2 hours. The plate is washed again 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 by a microtiter plate reader.
A standard curve is constructed using serial dilutions of control samples and the polypeptide concentration is plotted on the X-axis (log scale) and the fluorescence or absorbance is plotted on the Y-axis (uniform scale). A standard curve is used to interpolate the concentration of polypeptide in the sample.

Example 23: Formulation The present invention also provides a disease or disorder (eg, any one of the diseases or disorders disclosed herein) by administering to a subject an effective amount of a therapeutic agent. As above)
A method of treating and / or preventing is provided. Polynucleotides or polypeptides of the present invention (including fragments and variants), agonists or antagonists thereof, and / or antibodies thereto in combination with a therapeutic agent in a pharmaceutically acceptable carrier form (eg, a sterile carrier). Is meant.

Therapeutic agents will take into account the clinical condition of the individual patient (particularly the side effects of treatment with the therapeutic agent alone), the site of delivery, the mode of administration, the dosing regimen and other factors known to those of skill in the art, and the medical practice ( Formulated and dosed in a manner that adheres to the good medical practice. Therefore, the "effective amount" intended in the present specification is determined by taking such a consideration into consideration.

As a general suggestion, the total pharmaceutically effective amount of therapeutic agent administered parenterally per dose is in the range of about 1 μg / kg / day to 10 mg / kg / day of patient body weight,
This is at therapeutic discretion, as described above. More preferably, for the 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 humans. In the case of continuous administration, the therapeutic agent is typically about 1 μg / kg / hour to about 50 μg / kg.
Administered by either 1 to 4 injections per day 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 required to observe changes and the post-treatment interval at which a response occurs is:
It seems to change depending on the desired effect.

The therapeutic agent is administered orally, rectally, parenterally, intracistemally.
y), vaginal, intraperitoneal, topical (such as by powder, ointment, gel, drops, or transdermal patch), buccal or oral or nasal spray. "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. As used herein, the term "
“Parenteral” refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

The therapeutic agents of the invention are also suitably administered by a sustained release system. Suitable examples of sustained release therapeutic agents are oral, rectal, parenteral, vaginal, vaginal, intraperitoneal, topical (such as by powder, ointment, gel, drip or transdermal patch), Oral or oral or nasal spray. "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. As used herein, the term "parenteral" means intravenous, intramuscular,
Refers to modes of administration which include intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

The therapeutic agents of the invention are also suitably administered by a sustained release system. Suitable examples of sustained release therapeutic agents include suitable polymeric materials, such as semipermeable polymeric matrices in the form of shaped articles, eg films or microcapsules, suitable hydrophobic materials (
For example, as an emulsion in an acceptable oil) or ion exchange resins, and poorly soluble derivatives (such as poorly soluble salts).

Sustained-release matrices include polylactide (US Pat. No. 3,773,919).
No., EP 58,481), a copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers 22: 547-5.
56 (1983)), poly (2-hydroxyethyl methacrylate) (Lang)
er et al. Biomed. Mater. Res. 15: 167-277 (19
81), and Langer, Chem. Tech. 12: 98-105 (19
82)), ethylene vinyl acetate (Langer et al., Ibid.) Or poly-D.
-(-)-3-hydroxybutyric acid (EP133,988) is mentioned.

Sustained-release therapeutic agents also include therapeutic agents of the present invention encapsulated in liposomes (generally, Langer, Science 249: 1527-1533 (199).
0); Treat et al., Liposomes in the Therapy o.
f Infectious Disease and Cancer, Lope
z-Berstein and Fidler (ed.), Liss, New Yor
k, pages 317-327 and 353-365 (1989)). Liposomes containing therapeutic agents are prepared by methods known per se: D
E3,218,121; Epstein et al., Proc. Natl. Acad. S
ci. (USA) 82: 3688-3692 (1985); Hwang et al., Pr.
oc. Natl. Acad. Sci. (USA) 77: 4030-4034 (1
980); EP52,322; EP36,676; EP88,046; EP14.
3,949; EP 142,641; Japanese Patent Application No. 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP.
No. 102,324. Usually, the liposomes are small (about 200-800 Å) unilamellar type, where the lipid content is greater than about 30 mol% cholesterol and the selected proportion is adjusted for the optimal therapeutic agent. ..

In yet a further embodiment, the therapeutic agents of the invention are delivered by a pump (Langer, supra; Sefton, CRC Crit. Ref. Biom.
ed. Eng. 14: 201 (1987); Buchwald et al., Surger.
y 88: 507 (1980); Saudek et al., N. et al. Engl. J. Med.
321: 574 (1989)).

Another controlled release system is the Langer (Science 249: 1527-15).
33 (1990)).

For parenteral administration, in one embodiment, the therapeutic agent will generally be administered to the recipient in a desired degree of purity and in a pharmaceutically acceptable carrier, ie, the dosage and concentration employed. Formulated by mixing in a unit dosage injectable form (solution, suspension or emulsion) with a non-toxic and compatible with the other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds known to be deleterious to therapeutic agents.

Generally, the formulations are prepared by contacting the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, in a uniform and intimate contact. The product is then shaped, if desired, 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.

The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are not toxic to the recipients at the dosages and concentrations used, and include such substances as phosphates, citrates, succinates, acetic acid and other organic acids or their salts. Buffers such as; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides (eg, polyarginine or tripeptides); proteins such as serum albumin, gelatin or immunoglobulins; polyvinylpyrrolidone. Hydrophilic polymers such as; glycine, glutamic acid, aspartic acid or amino acids such as arginine; cellulose or its derivatives, mono-, di-, and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA ; Sugar sugars such as mannitol or sorbitol Lumpur; counterions such as sodium; and / or polysorbate include nonionic surfactants such as poloxamers or PEG.

The therapeutic agent is typically about 0.1 mg / ml to 100 mg / ml, preferably 1
It is formulated in such a vehicle at a concentration of -10 mg / ml and a pH of about 3-8. It is understood that the use of the particular excipients, carriers or stabilizers described above will result in the formation of polypeptide salts.

Any pharmaceutical agent used for therapeutic administration can be sterile. Sterility is easily achieved by filtration through a sterile filtration membrane (eg 0.2 micron membrane). Generally, the therapeutic agent is placed in a container having a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle.

Therapeutic agents typically are stored in unit-dose or multi-dose containers, such as sealed ampoules or vials, as an aqueous solution or as 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 the aqueous therapeutic agent solution and freeze-dry the resulting mixture. The lyophilized therapeutic agent is reconstituted with bacteriostatic water for injection to prepare an infusion solution.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the therapeutic agents of the 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, which notice may include government notices regarding manufacture, use, or sale for human administration. Indicates approval by the institution. In addition, therapeutic agents may be used in combination with other therapeutic compounds.

The therapeutic agents of the invention can be administered alone or in combination with an adjuvant. Adjuvants that may be administered with the therapeutic agents of the present invention include, but are not limited to: alum, alum + deoxycholic acid (I
mmunoAg), MTP-PE (Biocine Corp.), QS21 (
Genentech, Inc. ), BCG, and MPL. In certain embodiments, therapeutic agents of the invention are administered in combination with alum. In another specific embodiment, the Therapeutic Agents of the invention are administered in combination with QS-21. Additional adjuvants that may be administered with the therapeutic agents of the present invention include, but are not limited to, monophosphoryl lipid immunomodulators, AdjuV.
ax 100a, QS-21, QS-18, CRL1005, aluminum salt,
MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the therapeutic agents of the invention include, but are not limited to, MMR (measles, mumps, rubella), polio, chickenpox, tetanus / diphtheria, A. Hepatitis B, hepatitis B, influenza B, whooping cough (who
open cough), pneumonia, influenza, Lyme disease, rotavirus,
Cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,
Vaccines directed against protection against typhoid fever and pertussis. The combination can be administered either, for example, simultaneously as a mixture, separately but simultaneously or in parallel; or sequentially. This includes the description that the combined agents are administered together as a therapeutic mixture, and the procedure in which the combined agents are administered separately but simultaneously (eg, to the same individual via separate intravenous lines) is also Including. "Combination" administration further comprises the separate administration of one of the compounds or agents given first, followed by the second.

The therapeutic agents of the present invention can be administered alone or in combination with other therapeutic agents. Therapeutic agents that may 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 can be administered either, for example, simultaneously as a mixture, separately but simultaneously or in parallel; or sequentially. This includes the description that the combined agents are administered together as a therapeutic mixture, and also for procedures where the combined agents are administered separately but simultaneously (eg, to the same individual via separate intravenous lines). Also includes. “Combination” administration refers to one of the compounds or agents given first, followed by second.
Further comprising administering the two separately.

In one embodiment, the Therapeutics of the invention are administered in combination with members of the TNF family. TNF, TNF that can be administered with the therapeutic agents of the invention
Related or TNF-like molecules include, but are not limited to: soluble forms of TNF-α, lymphotoxin-α, (LT-α, TNF-β.
, Also known as), LT-β (found in the complex heterotrimer LT-α2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1B.
BL, DcR3, OX40L, TNF-γ (International Publication Number WO96 / 14328
), AIM-I (International Publication No. WO97 / 33899), endokine (end)
kine) -α (international publication number WO98 / 07880), TR6 (international publication number WO98 / 30694), OPG, and neutrokine (neutrok).
ine) -α (International Publication No. WO98 / 18921), OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD4.
0 and 4-IBB, TR2 (International Publication Number WO96 / 34095), DR3 (
International publication number WO97 / 33904), DR4 (International publication number WO98 / 328)
56), TR5 (International Publication Number WO98 / 30693), TR6 (International Publication Number WO98 / 30694), TR7 (International Publication Number WO98 / 41629), TR
ANK, TR9 (International Publication Number WO98 / 56892), TR10 (International Publication Number WO98 / 54202), 312C2 (International Publication Number WO98 / 06842)
, And TR12, and soluble forms of CD154, CD70, and CD1
53.

In certain embodiments, Therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and / or protease inhibitors. Nucleoside reverse transcriptase inhibitors that can be administered in combination with the therapeutic agents of the present invention include:
Including but not limited to: RETROVIR ^TM (zidovudine / AZT), VIDEX ^TM (didanosine / ddI), HIVID ^TM (zalcitabine / ddC), ZERIT ^TM (stavudine / d4T), EPIVIR ^{T M} (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, VIRA.
MUNE ^™ (nevirapine), RESCRIPTOR ^™ (delavirdine), and SUSTIVA ^™ (efavirens). Protease inhibitors that may be administered in combination with the therapeutic agents of the present invention include, but are not limited to, CRIXIVAN ^™ (indinavir).
), NORVIR ^™ (ritonavir), INVIRAS
E ^™ (saquinavir), and VIRACCEPT ^™ (nelfinavir). In certain embodiments, an antiretroviral agent, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, and / or protease inhibitor is used to treat AIDS and / or prevent or treat HIV infection. It may be used in any combination with the therapeutic agents of the invention.

[0854]   In another embodiment, the therapeutic agents of the invention are combined with an anti-opportunistic infection agent.
Can be administered. Anti-opportunistic infection drug that can be administered in combination with the therapeutic agent of the present invention
Agents include, but are not limited to, TRIMETHOP.
RIM-SULFAMETHOXAZOLE^TM, DAPSONE^TM, PEN
TAMIDINE^TM, ATOVAQUONE^TM, ISONIAZID^TM,
RIFAMPIN^TM, PYRAZINA MIDE^TM, ETHAMBUTOL ^TM , RIFABUTIN^TM, CLARITHROMYCIN^TM, AZIT
HROMYCIN^TM, GANCICLOVIR^TM, FOSCARNET^TM , CIDOFOVIR^TM, FLUCONAZOLE^TM, ITRACONAZ
OLE^TM, KETO CONAZOLE^TM, ACYCLOVIR^TM, FAM
CICOLVIR^TM, PYRIMETHAMINE^TM, LEUCOVORI
N^TM, NEUPOGEN^TM(Filgrastim /
G-CSF), and LEUKINE^TM(Sargrammostin
ostim) / GM-CSF). In certain embodiments, the therapeutic agents of the invention are
Opportunistic treatment of Pneumocystis carinii pneumonia
To prevent TRIMETHOPRIM-SULFAMETHOXAZO
LE^TM, DAPSONE^TM, PENTAMIDINE^TM, And / or
ATOVAQUONE^TMUsed in any combination with. Another specific implementation
In form, the therapeutic agent of the invention is an opportunistic Mycobacterium avi
In order to prophylactically treat or prevent um complex infection, ISONIAZID^TM,
RIFAMPIN^TM, PYRAZINA MIDE^TM, And / or ETH
AMBATOL^TMUsed in any combination with. In another particular embodiment
The therapeutic agent of the present invention is an opportunistic Mycobacterium tuberc.
RIFABUTIN for the prophylactic treatment or prevention of ulosis infection^{T M} , CLARITHROMYCIN^TM, And / or AZITROMYC
IN^TMUsed in any combination with. In another particular embodiment, the book
The therapeutic agents of the invention prophylactically treat or prevent opportunistic cytomegalovirus infections
In order to^TM, FOSCARNET^TM, And / or
Or CIDOFOVIR^TMUsed in any combination with. Another particular fruit
In embodiments, the therapeutic agents of the present invention prophylactically treat or prevent opportunistic fungal infections.
In order to do,^TM, ITRACONAZOLE^TM,
And / or KETO CONAZOLE^TMUsed in any combination with
. In another particular embodiment, the therapeutic agents of the invention are opportunistic herpes simplex virus.
ACY for prophylactically treating or preventing type I and / or type II infections
CLOVIR^TMAnd / or FAMCICOLVIR^TMAny combination with
Used in combination. In another particular embodiment, the therapeutic agents of the invention are opportunistic T
In order to prophylactically treat or prevent an oxoplasma gondii infection,
PYRIMETHAMINE^TMAnd / or LEUCOVORIN^TMWith
Used in any combination. In another specific embodiment, the therapeutic agent of the present invention
LEUCOVORI for the prophylactic treatment or prevention of opportunistic bacterial infections.
N^TMAnd / or NEUPOGEN^TMUsed in any combination with
.

In a further embodiment, the Therapeutic Agents of the invention are administered in combination with antiviral agents. Antiviral agents that may be administered with the therapeutic agents of the present invention include acyclovir, ribavirin, amantadine, and remantidine (remantidine).
Tidine), but is not limited thereto.

In a further embodiment, the Therapeutics of the invention are administered with an antibiotic. Antibiotics that can be administered with the therapeutic agent of the present invention include amoxicillin, β
-Lactamase, aminoglycoside, β-lactam (glycopeptide), β-lactamase, clindamycin, chloramphenicol, cephalosporin, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolone, macrolide, metronidazole, Penicillins, quinolones, rifampins, streptomycin, sulfonamides, tetracyclines, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin are not limited to these.

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

In certain embodiments, Therapeutics of the invention may be administered in combination with immunosuppressive agents. Immunosuppressant preparations that can be administered with the therapeutic agents of the invention include ORTHO
CLONE ^™ (OKT3), SANDIMMUNE ^™ / NEORAL ^™ /
SANGDYA ^™ (cyclosporine), PROGRAF ^™ (tacrolimus)
, CELLCEPT ^™ (microphenolate), azathioprine, glycoticosteroids, and RAPAMUNE ^™ (sirolimus). In certain embodiments, immunosuppressive agents may be used to prevent organ or bone marrow transplant rejection.

[0859]   In a further embodiment, the therapeutic agents of the invention are alone or in one or more intravenous immunizations.
It is administered in combination with an epiglobulin preparation. In combination with the therapeutic agent of the present invention
Intravenous immunoglobulin preparations that can be administered include GAMMAR^TM, IVE
EGAM^TM, SANDOGLOBULIN^TM, GAMMAGARD S / D ^TM And GAMIMUNE^TMBut is not limited to these. specific
In an embodiment of the present invention, the therapeutic agent of the present invention is suitable for transplantation treatment (eg, bone marrow transplantation).
Administered in combination with an intravenous immunoglobulin preparation.

In a further embodiment, the Therapeutics of the invention are administered alone or in combination with anti-inflammatory agents. Anti-inflammatory agents that can be administered with the therapeutic agents of the present invention include glucocorticoid and non-steroidal anti-inflammatory agents, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazole, pyrazolone. , Salicylic acid derivatives, thiazinecarboxamide, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzidoamine, bucolome, diphenpyramide, ditazole, emorfazone, guaiazulene. , Nabumetone, nimesulide, orgothein, oxaseprol, paraniline, perizoxal, pifoxime, prochiazone, proxazole, and tenidap. But, but it is not limited to these.

In another embodiment, the compositions of this invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may 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, phloxuridine (flo
xuridine), 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, diethylstilbestol diphosphate, chlorotrianisene, and test lactone); nitrogen mustard derivatives (Eg mephalen, chlorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (eg sodium betamethasone phosphate); and others (eg dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate,
And etoposide).

In a specific embodiment, the Therapeutics of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and predinnisone) or in any combination of the components of CHOP. In another embodiment, the Therapeutic Agents of the invention are administered in combination with Rituximab. In a further embodiment, the therapeutic agents of the invention are Rituxmab and CHOP.
Or with any combination of Rituxmab and CHOP components.

In a further embodiment, Therapeutics of the invention are administered in combination with cytokines. IL2 is a cytokine that can be administered together with the therapeutic agent of the present invention.
, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13,
Examples include, but are not limited to, IL15, anti-CD40, CD40L, IFN-γ and TNF-α. In another embodiment, the therapeutic agent of the present invention comprises any interleukin (IL-1α, IL-1β, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11
, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17
, IL-18, IL-19, IL-20 and IL-21).

In a further embodiment, Therapeutics of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that can be administered with the therapeutic agents of the present invention include Glioma Derived Growth Factor (GD) as disclosed in European Patent EP-399816.
GF); platelet-derived growth factor as disclosed in EP-682110-
A (PDGF-A); Platelet Induced Growth Factor-B (PDGF-B) as disclosed in European Patent EP-283217; Placental Growth Factor (PIGF) as disclosed in International Publication No. WO 92/06194; Hauser et al., Gorwhth Fa
placental growth factor-2 (PIGF-2) as disclosed in Ctors, 4: 259-268 (1993); vascular endothelial growth factor (VEGF) as disclosed in WO 90/13649; European Patent Vascular Endothelial Growth Factor-A (VEGF-A) as disclosed in EP-506477; International Publication No. WO 96/3951.
Vascular Endothelial Growth Factor-2 (VEGF-2); Vascular Endothelial Growth Factor-B (VEGF-3); Vascular Endothelial Growth Factor B as disclosed in International Publication No. WO 96/26736. -186 (VEGF-B186); International Publication Number WO
Vascular Endothelial Growth Factor-D (VEGF-D) as disclosed in 98/02543.
Vascular endothelial growth factor as disclosed in International Publication No. WO98 / 07832-
D (VEGF-D); and vascular endothelial growth factor-E (VEGF-E) as disclosed in German Patent DE19639601, but is not limited thereto. The above references are incorporated herein by reference.

In a further embodiment, the Therapeutics of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that can be administered with the therapeutic agents of the present invention include LEU
KINE ^™ (SARGRAMOSTIM ^™ ) and NEUPOGEN ^™ (
FILGRASTIM ^™ ), but is not limited thereto.

In a further embodiment, the Therapeutic Agents of the invention are administered in combination with fibroblast growth factor. Fibroblast growth factors that can be administered with the therapeutic agent of the present invention include FGF-1, FGF-2, FGF-3, FGF-4, FGF-5 and FGF.
-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, F
GF-12, FGF-13, FGF-14, and FGF-15 include, but are not limited to.

In a further embodiment, the Therapeutics of the invention are administered in combination with other therapeutic or prophylactic regimes (eg, radiation therapy).

Example 24: Method for Treating Decreased Levels of Polypeptides The present invention relates to a method for treating an individual in need of increasing levels of a polypeptide of the invention in the body, which method comprises: Administering to such an individual a composition comprising a therapeutically effective amount of an agonist of the invention (including a polypeptide of the invention). Further, it is understood that conditions caused by reduced normal or normal expression levels of secreted proteins in an individual can be treated by administering the polypeptides of the invention, preferably in secreted form. Thus, the invention also provides a method of treating an individual in need of increased levels of polypeptide. The method includes the step of administering to such an individual a therapeutic agent comprising an amount of the polypeptide that increases the activity level of the polypeptide in such individual.

For example, a patient with reduced levels of a polypeptide receives the polypeptide at a daily dose of 0.1-100 μg / kg for 6 consecutive days. Preferably the polypeptide is in secreted form. Exact details of dosing and formulation-based regimens are given in Example 2.
It is provided in 3.

Example 25: Method of Treating Elevated Levels of Polypeptides The present invention also relates to a method for treating an individual in need of decreasing levels of a polypeptide of the invention in the body, which method comprises: , Administering a composition comprising a therapeutically effective amount of an antagonist of the invention (including the polypeptides and antibodies of the invention) to such an individual.

In one example, antisense technology is used to inhibit production of a polypeptide of the invention. This technique is one example of how to reduce the level of a polypeptide, preferably a secreted form of the polypeptide, that results from various etiologies such as cancer.
For example, in patients diagnosed with abnormally elevated levels of polypeptide, antisense polynucleotides are administered at 0.5, 1.0, 1.5, 2.0 and 3.0 mg /
It is administered intravenously at kg / day for 21 days. If you are well tolerated of this procedure,
This treatment is repeated after a 7 day washout period. Formulations of antisense polynucleotides are provided in Example 23.

Example 26 Treatment Method Using Gene Therapy-Ex Vivo One method of gene therapy is to transplant fibroblasts capable of expressing a polypeptide into a patient. Fibroblasts are generally obtained from a subject by skin biopsy. The resulting tissue is placed in tissue culture medium and divided into small pieces. The nodule of tissue is placed on the wet surface of the tissue culture flask and approximately 10 pieces are placed in each flask. The flask is inverted and tightly closed, then left overnight at room temperature. After 24 hours at room temperature, the flask was inverted, the tissue mass was left fixed at the bottom of the flask and fresh medium (eg Ha containing 10% FBS, penicillin and streptomycin) was used.
m F12 medium) is added. The flask is then incubated at 37 ° C for approximately 1 week.

At this point, fresh medium is added and then replaced every few days. After culturing for another two weeks, a monolayer of fibroblasts appears. Trypsinizing this monolayer,
Scale up to a larger flask.

PMV-7 (Kirsc) flanked by long terminal repeats of Moloney murine sarcoma virus.
hmeier, P.M. 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.

The cDNAs encoding the polypeptides of the present invention were prepared as described in Example 1 using primers and with appropriate restriction sites and start / stop codons, respectively, and the 5'and 3'terminal sequences, respectively. 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 backbone and the amplified EcoRI and HindIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under suitable conditions for ligating the two fragments. The ligation mixture is then used to transform bacterial HB101. It is then plated on agar containing kanamycin to confirm that the vector has the gene of interest inserted correctly.

Amphotropic pA317 or GP + am12 packaging cells were
Grow to confluent density in tissue culture in Dulbecco's Modified Eagle Medium (DMEM) with 0% Calf Serum (CS), Penicillin and Streptomycin. The MSV vector containing the gene of interest is then added to the medium and the packaging cells are transduced with this vector. At this time, the packaging cells produce infectious viral particles containing the gene (herein, the packaging cells are referred to as producer cells).

Fresh medium is added to the transduced producer cells and this medium is then harvested from 10 cm plates of confluent producer cells. The spent medium containing infectious viral particles is filtered through a Millipore filter to remove detached producer cells and then used to infect fibroblasts. Media is removed from the subconfluent plates of fibroblasts and quickly replaced with media from producer cells. This medium is removed and replaced with fresh medium. If the virus titer is high, virtually all fibroblasts will be infected and no selection is required. 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 the protein is being produced.

The engineered fibroblasts are then transplanted into the 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 the use of endogenous polynucleotide sequences of the invention, eg, in US Pat. No. 5,641,670 (issued June 24, 1997);
International Publication Number WO 96/29411 (Published September 26, 1996); International Publication Number WO 94/12650 (Published August 4, 1994); Koller et al., P.
roc. Natl. Acad. Sci. USA, 86: 8932-8935 (1
989); and Zijlstra et al., Nature, 342: 435-438.
Operably linked to the promoter via homologous recombination, as described in (1989). The method involves activation of a gene that is present in the target cell but is not expressed in the cell or is expressed at a lower level than desired.

A polynucleotide construct containing a promoter and targeting sequence is made. This target sequence is homologous to the 5'non-coding sequence of the endogenous polynucleotide sequence, which flanks 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 upon homologous recombination. PCR of this promoter and targeting sequence
Can be used to amplify. Preferably, the amplified promoter contains different restriction enzyme sites on the 5'and 3'ends. Preferably, the 3'end of the first targeting sequence contains the same restriction enzyme sites as the 5'end of the amplified promoter, and the 5'end of the second targeting sequence is the 3'end of the amplified promoter. Contains the same restriction sites as.

The amplified promoter and amplified targeting sequence are digested with the appropriate restriction enzymes, followed by treatment with calf intestinal phosphatase. The digested promoter and digested targeting sequence are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for ligation of these two fragments. The construct is size fractionated on an agarose gel, 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 may also be administered with a transfection facilitating agent such as liposomes, viral sequences, viral particles, precipitating agents and the like. Such methods of delivery are known in the art.

Once the cells are transfected, homologous recombination will occur and the promoter will be operably linked to the endogenous polynucleotide sequence. This results in expression of the polynucleotide corresponding to the polynucleotide in the cell. Expression can be detected by immunological staining or any other method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The obtained tissue is DMEM +
Place in 10% fetal bovine serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the surface of the plastic with nutrient medium. An aliquot of cell suspension is removed for counting and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is mixed 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 aspirated, and the cells resuspended in electroporation buffer containing 1 mg / ml acetylated bovine serum albumin. This final cell suspension contains approximately 3 × 10 ⁶ cells / ml. Electroporation should be performed immediately after resuspension.

Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus corresponding to a polynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst, NY) was constructed.
) Is digested with HindIII. The CMV promoter is amplified by PCR with an XbaI site at the 5'end and a BamHI site at the 3'end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a HindIII site at the 5'end and an XbaI site at the 3'end; the other non-coding sequence (fragment). 2) is amplified with a BamHI site at the 5'end and a HindIII site at the 3'end. The CMV promoter and fragments thereof (1 and 2) were cloned into the appropriate enzymes (CMV promoter-XbaI and BamHI; fragment 1-XbaI; fragment 2-B.
amHI) and digested together. The resulting ligation product was HindII
I digested and ligated with HindIII digested pUC18 plasmid.

Plasmid DNA was added to a sterile cuvette (B
io-Rad). Final DNA concentration is generally at least 120μ
g / ml. Then 0.5 ml of the cell suspension (containing approximately 1.5 × 10 ⁶ cells) is added to the cuvette and the cell suspension and DNA solution are gently mixed. Electroporation is performed using a Gene-Pulser instrument (Bio-Rad). The capacitance and voltage are set to 960 μF and 250-300 V, respectively. Increasing the voltage reduces cell survival, but dramatically increases the proportion of viable cells that stably integrate the introduced DNA into their genome. Given these parameters, the pulse time is about 14-2
0 mSec should be observed.

The electroporated cells are kept at room temperature for about 5 minutes, then the contents of the cuvette are gently removed using a sterile transfer pipette. The cells were placed in a 10 cm dish containing pre-warmed nutrient medium (D containing 15% bovine serum).
MEM) 10 ml directly 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.

The engineered fibroblasts are then injected into the host, either alone or after being grown to confluence on cytodex 3 microcarrier beads. To do. here,
The fibroblasts produce the protein product. The fibroblasts can then be introduced into a patient as described above.

Example 28: Treatment Methods Using Gene Therapy-In Vivo Another aspect of the invention is the use of in vivo gene therapy methods to treat disorders, diseases and conditions. This method of gene therapy involves the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease expression of the polypeptide. The polynucleotide of the invention may 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, eg, WO 90/11.
092, WO98 / 11779; U.S. Pat. Nos. 5,693,622, 5,705,51.
51, ibid. 5580859; Tabata et al., Cardiovasc. Re
s. 35 (3): 470-479 (1997); Chao et al., Pharmaco.
l. Res. 35 (6): 517-522 (1997); Wolff, Neur.
omuscul. Disord. 7 (5): 314-318 (1997), Sc.
hwartz et al., Gene Ther. 3 (5): 405-411 (1996).
Tsurumi et al., Circulation 94 (12): 3281-32.
90 (1996), incorporated herein by reference.

The polynucleotide construct is delivered by any method that delivers an injectable substance to cells of an animal, such as injection into the interstitial space of a 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 lipofectin that acts to assist, facilitate, or facilitate entry into cells. (Including a precipitating agent) is not included. However, the polynucleotides of the invention can also be prepared in liposome formulations (eg, Felgner) that can be prepared by methods well known to those of skill in the art.
P. L. (1995) Ann. NY Acad. Sci. 772: 126-1
39 and Abdallah B. (1995) Biol. Cell 85 (
1): 1-7)).

The polynucleotide vector construct used in this method of gene therapy is
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 the expression of 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 for the production of desired polypeptides for periods of up to 6 months.

This polynucleotide construct can be used in tissues (muscle, skin, brain, lung, liver,
(Including spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue) It can be delivered to the space. The interstitial spaces of this tissue are intercellular fluids, mucopolysaccharide substrates (between reticulum 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 within natural muscle cells or in the hiatus of bone. This is likewise the space occupied by circulating plasma and lymphatic fluid of the lymphatic channels. Delivery of muscle tissue to the interstitial space is preferred for the 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, which delivery and expression can be effected on undifferentiated cells or cells that are not fully differentiated (eg, blood stem cells). Or skin fibroblasts). In vivo, muscle cells are particularly competent for their ability to take up and express polynucleotides.

For naked polynucleotide injections, an effective dosage of DNA or RNA is in the range of about 0.05 g / kg body weight to about 50 mg / kg body weight. Preferably, this dosage is about 0.005 mg / kg to about 20 mg / kg, and more preferably 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. Suitable and effective dosages of nucleic acid sequences can be readily determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used,
This includes, for example, inhalation of aerosol formulations, especially for delivery to the mucosa of the lungs or bronchial tissues, throat, or nose. In addition, the naked polynucleotide construct can be delivered to the artery during the angioplasty procedure by the catheter used in this procedure.

The dose response effect of infused polynucleotides on muscle in vivo is determined as follows. Appropriate template DNA for the production of mRNA encoding the polypeptide of the present invention is prepared according to standard recombinant DNA methodologies. Mold DN
Either A, which can be either circular or linear, is used as naked DNA or is complexed with liposomes. The mouse quadriceps is then injected with varying amounts of template DNA.

To 5-6 week old female and male Balb / C mice, 0.3 ml of 2.5%
Anesthetize by intraperitoneal injection of Avertin. A 1.5 cm incision is made in the anterior thigh and the quadriceps are visualized directly. Template DNA is placed in a 0.1 ml carrier and injected with a 1 cc syringe through a 27 gauge needle for 1 minute into the knee 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 localization and the skin is closed with stainless steel clips.

After a suitable incubation time (eg 7 days), muscle extracts are prepared by cutting out whole quads. Every 5th 15 μm section of an individual quadriceps is histochemically stained for protein expression. The time course for protein expression can be done in a similar fashion except that four animals from different mice are harvested at different times. Persistence of DNA in muscle after injection can be determined by Southern blot analysis after preparation of total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experiments in mice are shown as naked D
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, miniature pig, goat,
Animals of any species, including but not limited to sheep, cows and non-human primates such as baboons, monkeys and chimpanzees, can be used to produce transgenic animals. In certain embodiments, 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.

Any technique known in the art may be used to introduce a transgene (ie, a polynucleotide of the invention) into an animal to establish the foo of the transgenic animal.
nder line) can be generated. Such techniques are described in Pronuclear Microinjection (Patterson et al., Appl. Microbiol. Biotec.
hnol. 40: 691-698 (1994); Carver et al., Biotec.
hology (NY) 11: 1263-1270 (1993); Wright
Et al., Biotechnology (NY) 9: 830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); Germline retrovirus-mediated gene transfer (Van der Putten et al., Pr.
oc. Natl. Acad. Sci. USA 82: 6148-6152 (19).
85)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompso)
n et al., Cell 56: 313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3: 180).
3-1814 (1983)); introduction of the polynucleotide of the present invention using a gene gun (see, for example, Ulmer et al., Science 259: 1745 (1993)); to embryonic pluripotent stem cells. Introduction of the nucleic acid construct and transfer of this stem cell into the blastocyst; and sperm-mediated gene transfer (La
Vitrano et al., Cell 57: 717-723 (1989)); and the like, but are not limited thereto. For a review of such techniques, see Gordon, "Transgenic An," which is incorporated herein by reference in its entirety.
imals ", Intl. Rev. Cytol. 115: 171-229 (19
89).

Any technique known in the art can be used to generate transgenic clones containing a polynucleotide of the invention (eg, of cultured, quiescent-induced embryonic cells, fetal cells or adult cells). Nuclear transfer of cell-derived enucleated oocytes into the nucleus (Campell et al., Nature 380: 64-66 (1996); Wil
mut et al., Nature 385: 810-813 (1997))).

The present invention provides transgenic animals having the transgene in all their cells, as well as animals having the transgene in some (but not all) of the cells (ie, mosaic animals or chimeras). To do. The transgene may be as a single transgene or in a concatemer (eg head-to-head tandem or head-to-head).
Multiple copies, such as tail tandem type). This transgene is also described, for example, in the teaching of Lasko et al. (Lasko et al., Proc. Natl. Ac.
ad. Sci. USA 89: 6232-6236 (1992)) and can be selectively introduced into and activated by specific cell types. The regulatory sequences required for such cell type-specific activation depend on the particular cell type of interest, and they will be apparent to those of skill in the art. Targeting of the gene is preferred when it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene. Briefly, if such a technique is utilized, a vector containing several nucleotide sequences homologous to the endogenous gene may be used to bind the nucleotide sequence of the endogenous gene via homologous recombination with the chromosomal sequence. And is designed for the purpose of destroying the function of the nucleotide sequence. Transgenes can also be selectively introduced into specific cell types, and thus, for example, Gu et al. (Gu et al. Science 26
5: 103-106 (1994)), the endogenous gene is inactivated only in the introduced cell type. The regulatory sequences required for such cell type-specific inactivation depend on the particular cell type of interest, and they will be apparent to those of skill in the art.

Once a transgenic animal is produced, expression of the recombinant gene can be assayed using standard techniques. Initial screening can be accomplished by Southern blot analysis or PCR techniques to confirm that transgene integration has occurred by analysis of animal tissue. The level of transgene mRNA expression in the tissues of transgenic animals was also determined by Northern blot analysis, in situ hybridization analysis and reverse transcriptase P of tissue samples obtained from the animals.
It can be assessed using techniques including, but not limited to CR (rt-PCR). Samples of transgenic gene expressing tissues can also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once founder animals are produced, they can be crossed, inbred, outbred or crossed to produce colonies of particular animals. Examples of such mating strategies include, but are not limited to: Founder outcrosses with more than one integration site to establish another lineage; additive expression of each transgene. By effect, inbreeding of separate strains to produce composite transgenics expressing higher levels of the transgene; a given routine for both enhancing expression and eliminating the need for screening animals by DNA analysis. Hybridization of heterozygous transgenic animals that produce animals homozygous for the site of integration; crossing of separate homozygous strains to generate complex heterozygous or homozygous lines; and to the desired experimental model Mating to place the transgene on a suitable different background.

The transgenic animals of the present invention may be used to describe the biological function of the polypeptides of the present invention, study conditions and / or disorders associated with aberrant expression, and ameliorate such conditions and / or disorders. It has applications including, but not limited to, animal model systems useful for screening for effective compounds.

Example 30: Knockout Animals Endogenous gene expression is also demonstrated using targeted homologous recombination for gene and / or gene expression.
Alternatively, it can be reduced by inactivating or “knocking out” its promoter (eg, Smithies et al., Nature 317: 230-2).
34 (1985); Thomas and Capecchi, Cell 51: 5.
03-512 (1987); Thompson et al., Cell 5: 313-32.
1 (1989); each of which is incorporated herein by reference in its entirety). For example, a variant of the invention, a non-functional polynucleotide (or completely unrelated DNA) flanked by DNA homologous to an endogenous polynucleotide sequence (either the coding or regulatory region of this gene). Sequence) can be used with or without a selectable marker and / or a negative selectable marker to transfect cells expressing a polypeptide of the invention in vivo. In another embodiment, techniques known in the art are used to make knockouts in cells that contain the gene of interest but do not express it. Insertion of this DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such an approach is particularly suited to the research and agricultural fields where modifications to embryonic stem cells may be used to produce animal progeny that have inactive targeted genes (eg, Thomas and Capecchi 1987 and Thompson 1989). , See above). However, this approach is conventional for use in humans when the recombinant DNA construct is directly administered or targeted to the required site in vivo using appropriate viral vectors, which will be apparent to those skilled in the art. Can be adapted to.

In a further embodiment of the invention, a cell genetically engineered to express a polypeptide of the invention, or a cell genetically engineered not to express a polypeptide of the invention (eg, a knockout), Administer to patients in vivo. Such cells can be obtained from patients (ie, animals, including humans) or MHC compatible donors, and can be fibroblasts, bone marrow cells, blood cells (eg lymphocytes), adipocytes, muscle cells, endothelial cells, etc. Can be included, but is not limited to. The cells can be transformed, for example, by transduction (using a viral vector and preferably a vector that integrates the transgene into the cell genome) or transfection procedures (plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Endogenous regulation to introduce a coding sequence of a polypeptide of the invention into a cell or to bind to this coding sequence and / or a polypeptide of the invention. To destroy the array
Genetically engineered in vitro using recombinant DNA technology. The coding sequence of the polypeptide of the 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 invention. Engineered cells that express and preferably secrete a polypeptide of the invention can be introduced systemically into the patient, eg, in 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 Patent) No. 5,399,349 and Mu
See Lligan and Wilson, US Pat. No. 5,460,959. Each of these is hereby incorporated by reference in its entirety).

If the cells to be administered are non-self or non-MHC compatible cells, they may be administered using well known techniques that interfere with the development of a host immune response against this transduced cell. 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 permitting exchange of components with the immediate extracellular environment.

The transgenic and "knockout" animals of the present invention may be used to describe the biological function of the polypeptides of the present invention, to study diseases, disorders and / or conditions associated with aberrant expression, as well as to such diseases. , Having animal model systems useful in screening compounds effective in ameliorating disorders and / or conditions, but not limited thereto.

Example 31: Production of Antibodies (a. Hybridoma Technology) The antibodies of the invention can be prepared by a variety of methods. (Current Pr
otocols, 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 the polypeptide of the invention is prepared and purified to be substantially free of natural contaminants. Such preparations are then introduced into animals to produce greater specific activity of polyclonal antisera.

Monoclonal antibodies specific for the polypeptides of the present 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 .; Y.
, Pp. 563-681 (1981)). Generally, an animal, preferably a mouse, is immunized with a polypeptide of the invention, or more preferably with secretory polypeptide expressing cells. Such polypeptide-expressing cells are preferably supplemented with 10% fetal calf serum (inactivated at about 56 ° C.) in any suitable tissue culture medium, and about 10 g / l of non-essential amino acids, about 1 g / l. Cultured in Earle's modified Eagle's medium supplemented with 1,000 U / ml penicillin and about 100 μg / ml streptomycin.

The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be used in accordance with the present invention; however, AT
It is preferred to use the parental myeloma cell line (SP2O) available from CC. After fusion, the resulting hybridoma cells were selectively maintained in HAT medium and then Wands et al. (Gastroenterology 80: 225-232 (
Cloning by limiting dilution as described by 1981)). The hybridoma cells obtained by such selection are then assayed to identify clones secreting antibodies capable of binding the polypeptides of the invention.

Alternatively, additional antibodies capable of binding the polypeptides of the present invention may be generated in a two step procedure using anti-idiotypic antibodies. Such a method
Taking advantage of the fact that the antibody itself is the antigen and it is therefore possible to obtain an antibody which binds to the second antibody. According to this method, protein-specific antibodies are used to immunize animals, preferably mice. The splenocytes of such animals are then used to produce hybridoma cells. The hybridoma cells are then screened to identify clones producing antibodies that can be blocked by the polypeptides of the invention for their ability to bind to the polypeptides of the antibodies of the invention. Such antibodies include anti-idiotypic antibodies to antibody polypeptides specific for the proteins of the invention, and immunize animals to induce the formation of additional antibody polypeptide specific proteins of the invention. Used for.

For in vivo use of antibodies in humans, the antibody is "humanized." Such antibodies can be produced by using genetic constructs derived from hybridoma cells which produce the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and discussed herein (for a review, Morrison, Science 229: 120).
2 (1985); Oi et al., BioTechniques 4: 214 (1986).
); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi
Et al., EP 171496; Morrison et al., EP 173494; Neub.
erger et al., WO 8601533; Robinson et al., WO 87026.
71; Boulianne et al., Nature 312: 643 (1984); N.
See Euberger et al., Nature 314: 268 (1985)).

(B) Isolation of Antibody Fragments Directed to Polypeptides from a Library of scFvs The naturally-occurring V gene isolated from human PBL can be or can be exposed to a donor. Was constructed into a library of antibody fragments that contained a reactivity to a polypeptide of the invention that was not present (see, eg, US Pat. No. 5,885,793, which is incorporated herein by reference in its entirety). ).

Library rescue. A library of scFvs is constructed from RNA of human PBLs as described in PCT Publication WO 92/01047. To rescue the phage displaying the antibody fragment, approximately 10 ⁹ E. coli carrying the phagemid were rescued. 50 ml 2 × TY (containing 1% glucose and 100 μg / ml ampicillin) (2 × TY-AMP-GLU) was inoculated with E. coli and 0.8 OD with shaking. D. Grow to. 5 of this culture
50 ml of 2 × TY-AMP-GLU was inoculated with 2 ml of 2 × 10 8 TU Δ gene 3 helper (M13Δ gene III, PCT publication WO92 / 01047).
) Is added and the culture is incubated at 37 ° C. for 45 minutes without shaking, then at 37 ° C. for 45 minutes with shaking. This culture was incubated for 10 minutes at 4000 rpm. p. m. Centrifuge at rt and resuspend pellet in 2 liters of 2xTY containing 100 μg / ml ampicillin and 50 μg / ml kanamycin and grow overnight. PCT published WO
Prepare as described in 92/01047.

M13Δ gene III is prepared as follows: M13Δ gene III helper phage does not encode gene III protein. Therefore, phage displaying antibody fragments (phagemids) have greater binding avidity for antigen. During phage morphogenesis, infectious M13Δ gene III particles are made by growing helper phage in cells carrying the pUC19 derivative that supplies the wild-type gene III protein. The culture is incubated for 1 hour at 37 ° C without shaking and then for another hour at 37 ° C with shaking. Centrifuge the cells (IEC-Centra 8,400r
． p. m. For 10 minutes), 100 μg / ml ampicillin and 25 μg / ml
2xTY broth containing 2 ml of kanamycin (2xTY-AMP-KAN) 3
Resuspended in 00 ml and grown overnight at 37 ° C with shaking. Phage particles were purified and concentrated from the culture medium by two rounds of PEG precipitation (Sambrook et al., 1990), resuspended in 2 ml PBS, and passed through a 0.45 μm filter (Minisart NML; Sartorius) for approximately 10 ^1. A final concentration of ³ transducing units / ml (ampicillin resistant clones) is obtained.

Panning the library. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg / ml or 10 μg / ml of the polypeptides of the invention. Tubes are 3% with 2% Marvel-PBS
Block for 2 hours at 7 ° C, then wash 3 times in PBS. Approximately 10 ¹³ TU of phage are applied to the tubes and incubated for 30 minutes at room temperature with tumbling up and down on a turntable, then left to stand for a further 1.5 hours. The tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. The phage were eluted by adding 1 ml of 100 mM triethylamine and spinning up and down for 15 minutes on a turntable, then adding 0.5 ml of this solution to 1 ml.
． Immediately neutralize with 0 M Tris-HCl, pH 7.4. The eluted phage were then incubated with the bacteria for 30 minutes at 37 ° C. to allow 10 ml of mid-log phase E. E. coli TG1. Then E. E. coli is plated on TYE plates containing 1% glucose and 100 μg / ml ampicillin. The resulting bacterial library is then rescued with Δ gene 3 helper phage as described above to prepare phage for the next round of selection. This process is then followed by all four of the affinity purifications.
Repeat tube wash with PBS, 0.1% T for 3 and 4 times.
Increase 20-fold with ween-20 and 20-fold with PBS.

Characterization of binders. Phage eluted from the third and fourth round of selection were used to transform E. coli. E. coli HB2151 and soluble scFv are generated from a single colony for the assay (Marks et al., 1991). The ELISA is performed using microtiter plates coated with either 10 pg / ml of the polypeptide of the invention in 50 mM bicarbonate, pH 9.6. ELISA
PCR positive printing (for example, PCT publication WO
92/01047), and then further characterized by sequencing. These ELISA positive clones can also be cloned using techniques known in the art (eg, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody / antigen binding, and competitive agonist activity. Or competitive antagonist activity, etc.).

Example 32: Assay for Detecting Stimulation or Inhibition of B Cell Proliferation and Differentiation Generation of a functional humoral immune response was achieved between B lineage cells and their microenvironment.
It requires both soluble and cognate signaling. The signal can convey a positive stimulus (which causes cells of the B lineage to sustain programmed development) or a negative stimulus (which directs the cell to block the current-generating pathway). To date, IL-2, IL-4, IL-5, IL-6, IL-7, IL
A number of stimulatory and inhibitory signals have been found to influence B cell responsiveness, including -10, IL-13, IL-14 and IL-15. Interestingly, these signals, which are weak effectors in their own right, can be combined with various costimulatory proteins to induce activation, proliferation, differentiation, homing, resistance, and death in B cell populations.

One of the most studied classes of B cell costimulatory proteins is the TNF superfamily. Within this family, CD40, CD27 and CD30
It has been found to regulate a variety of immune responses with its respective ligands, CD154, CD70 and CD153. Assays that allow the detection and / or observation of proliferation and differentiation of these B cell populations and their progenitor cells 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 differentiation, proliferation, or inhibition of B cell populations and their precursors.

In Vitro Assay—Ability of the 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. Evaluate about. Activity of the polypeptides of the invention on purified human tonsillar B cells (qualitatively 0.1-10,000 n
(measured over a dose range of g / mL) is evaluated in a standard B lymphocyte costimulation assay. In this assay, purified tonsil B cells were used as a priming factor 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 cooperate with SAC and IgM cross-linking to induce B cell proliferation as measured by tritiated thymidine incorporation. New synergists can be easily identified using this assay. This assay involves the isolation of human tonsillar B cells by magnetic bead (MACS) depletion of CD3 positive cells. The resulting cell population is CD45R
Greater than 95% of B cells when assessed by expression of (B220).

Each sample at various dilutions was placed in an individual well of a 96-well plate, to which culture medium (10% FBS, 5 × 10 ⁻⁵ M 2ME, 100 U / m 2
1 penicillin, 10 μg / ml streptomycin, and 10 ⁻⁵ dilution of SA
10 ⁵ B cells in a total volume of 150 μl suspended in RPMI 1640 containing C) are added. Proliferation or inhibition started at 72 hours after addition of the factor, 3
Quantify with H-thymidine (6.7 Ci / mM) by 20 h pulse (1 μCi / well). Positive and negative controls are IL2 and medium, respectively.

In Vivo Assay-BALB / c mice are injected twice daily (intraperitoneally) with buffer alone, or 2 mg / kg of the polypeptides of the invention, or their shortened forms. 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 spleens treated with the polypeptide of the invention results in activity of this polypeptide on spleen cells, such as diffusion of periarterial lymphoid sheaths and / or red pulp regions. A significant increase in nucleated cellularity is noted, which may indicate activation of B cell population differentiation and proliferation. Anti-CD, a B cell marker
Using immunohistochemical studies with 45R (B220), any physiologic changes to spleen cells (eg spleen tissue disruption) invade the established T cell regions, a vaguely defined B cell compartment. Determine whether due to increased B cell presentation within.

Using flow cytometric analysis of spleens from mice treated with the polypeptide, the polypeptide is ThB +, CD45R (B220) dull B.
It is shown whether the ratio of cells is specifically increased over that observed in control mice.

Similarly, the putative outcome of in vivo presentation of increased mature B cells is serum I
g is the 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 the polypeptides of the invention.
However, one 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 polynucleotides or polypeptides of the invention.

Example 33 T Cell Proliferation Assay Proliferation Assay for Resting PBLs A CD3 induced proliferation assay is performed on PBMC and measured by ³ H-thymidine incorporation. The 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 and then wash 3 times with PBS. PBMCs were isolated from human peripheral blood by F / H gradient centrifugation, and various concentrations of TNFδ and / or TNF
In RPMI containing 10% FCS and P / S in the presence of ε protein, m
Add to quadruplicate wells (5 × 10 ⁴ / well) of Ab-coated plates (total volume 200 μl). The relevant protein buffer and media alone are controls. After 48 hours of incubation at 37 ° C., the plates were spun at 1000 rpm for 2 minutes and 100 μl of supernatant was removed, and IL-2 (or other cytokine) was added if an effect on proliferation was observed. Stored at -20 ° C for measurement.
The wells were supplemented with 100 μl of medium containing 0.5 μCi of ³ H-thymidine,
And it cultures at 37 degreeC for 18 to 24 hours. Wells were harvested 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 was used as TNFδ and / or
Or used as a negative control for the effect of TNFε protein.

Alternatively, proliferation assays on resting PBLs (peripheral blood lymphocytes) are measured by ³ H-thymidine incorporation. The assay is performed as follows. PBMCs from human peripheral blood were treated with Ficoll (LSM, ICN Biotech
isolated by gradient centrifugation at 10% (Fetal Calf Serum, Biofluids, Rock).
file, MD) / RPMI (Gibco BRL, Gaithersber
g, MD) overnight. This overnight incubation period
Adherent cells adhere to plastic. This gives a lower background in the assay. This is because there are few cells that can act as antigen-presenting cells or can produce growth factors. The next day, non-adherent cells are harvested, washed and used in the proliferation assay. The assay is performed in 96-well plates with 2 × 10 ⁴ cells / well in a final volume of 200 μl. Supernatants expressing the protein of interest (eg, CHO or 293T supernatant) were tested at a final dilution of 30%, thus 60 μl were added to 140 μl of 10% F containing cells.
Add to CS / RPMI. Control supernatants are used at the same final dilution and express the following proteins: vector (negative control), IL-2 ( ^* ),
IFNγ, TNFα, IL-10 and TR2. In addition to control supernatant, recombinant human IL-2 (R & D Systems, Minneapolis, MN)
Is also used at a final concentration of 100 ng / ml. After 24 hours of culture,
Pulse with Ci ³ H thymidine (Nen, Boston, MA). Cells are then harvested 20 hours after the pulse and ³ H thymidine incorporation is used as an indicator of proliferation. Results are expressed as the mean ± standard error of triplicate samples. ( ^* ) Control cytokine I produced in each transfection
The amount of L-2, IFNγ, TNFα and IL-10 was 300 pg-5 ng / m.
It varies between l.

Costimulation Assay Costimulation assays on resting PBLs (peripheral blood lymphocytes) are performed in the presence of antibodies immobilized against CD3 and CD28. The use of antibodies specific for the constant region of CD3 mimics the induction of T cell activity that occurs through stimulation of T cell receptors by antigen. TC in the absence of costimulatory signal (second signal)
Cross-linking of R (first signal) results in a very low induction of proliferation, and ultimately leads to a state of "anergy", which is characterized by lack of proliferation and inability to produce cytokines. Occurs. Addition of costimulatory signals, such as antibodies to CD28, that mimic the action of costimulatory molecules. B7-1 expressed on activated APCs results in enhanced T cell responses, including cell survival and IL-2 production. Thus, this type of assay can detect both positive and negative effects on T cell proliferation caused by the addition of supernatant expressing the protein of interest.

This assay is performed as follows. 100 ng / m in a final volume of 100 μl
l anti-CD3 and 5 μg / ml anti-CD28 (Pharmingen, San D
iego, CA) to coat 96-well plates and incubate overnight at 4 ° C. Wash plate twice with PBS before use. PBMCs from human peripheral blood were treated with Ficoll (LSM, ICN Biotechnology
s, Aurora, Ohio) isolated by gradient centrifugation and 10% FCS
(Fetal Calf Serum, Biofluids, Rockville
e, MD) / RPMI (Gibco BRL, Gaithersberg, MD)
) Incubate overnight. This overnight incubation period allows the adherent cells to adhere to the plastic. This gives a lower background in the assay. This is because there are few cells that can act as antigen-presenting cells or can produce growth factors. The next day, non-adherent cells are harvested, washed and used in the proliferation assay. This assay was run 2x1 in a final volume of 200 μl.
Performed in 96-well plates using the 0 ⁴ cells / well. Supernatants expressing the protein of interest (eg, CHO or 293T supernatant) were tested at a final dilution of 30%, thus 60 μl were added to 140 μl of 10% FCS / R containing cells.
Add to PMI. Control supernatants were used at the same final dilution and express the following proteins: vector only (negative control), IL-2, IFNγ,
TNFα, IL-10 and TR2. In addition to control supernatant, recombinant human I
L-2 (R & D Systems, Minneapolis, MN) is also used at a final concentration of 10 ng / ml. After 24 hours of culture, each well is pulsed with 1 μCi of ³ H thymidine (Nen, Boston, MA). Then pulse the cells 2
Harvested after 0 hours and ³ H thymidine incorporation is used as an indicator of proliferation.
Results are expressed as the mean ± standard error of triplicate samples.

Costimulation Assay: IFNγ and IL-2 ELISA This assay is performed as follows. 300 ng / m at a final volume of 500 μl
either 1 or 600 ng / ml anti-CD3 and 5 μg / ml anti-CD2
Coat 24-well plates with 8 (Pharmingen, San Diego, CA) and incubate overnight at 4 ° C. Wash plate twice with PBS before use. PBMC was isolated from human peripheral blood by Ficoll (LSM,
Isolated by ICN Biotechnologies, Aurora, Ohio) gradient centrifugation and 10% FCS (Fetal Calf Serum,
Biofluids, Rockville, MD) / RPMI (Gibco B
Culture overnight in RL, Gaithersberg, MD). This overnight incubation period allows the adherent cells to adhere to the plastic. This gives a lower background in the assay. This is because there are few cells that can act as antigen-presenting cells or can produce growth factors. The next day, non-adherent cells are harvested, washed and used in a co-stimulation assay. The assay is performed in pre-coated 24-well plates with 1 × 10 ⁵ cells / well in a final volume of 900 μl. The supernatant expressing the protein of interest (293T supernatant) is tested at a final dilution of 30%, so 300 μl is added to 600 μl of 10% FCS / RPMI containing cells. Control supernatants are used at the same final dilution and express the following proteins: vector only (
Negative control), IL-2, IFNγ, IL-12 and IL-18. In addition to control supernatant, recombinant human IL-2 (all cytokines
(Purchased from Systems, Minneapolis, MN) is also used at a final concentration of 10 ng / ml, IL-12 at a final concentration of 1 ng / ml and IL-18 at a final concentration of 50 ng / ml. Controls and unknown samples are tested in duplicate. Supernatant samples (250 μl) are collected 2 and 5 days after the start of the assay. R ELISA for testing for secretion of IFNγ and IL-2
& D Systems (Minneapolis, MN). Results are expressed as mean ± standard error of duplicate samples.

Proliferation Assay of Pre-Activated Resting T Cells On the cells previously activated with the lectin phytohemagglutinin (PHA), Proliferation assay is performed. Lectins are polymeric plant proteins that can bind to residues on T cell surface glycoproteins, including TCRs, and act as polyclonal activators. PBL
Are treated with PHA and then cultured in the presence of low doses of IL-2 mimic effector T cells. These cells are generally more sensitive to further activation induced by growth factors such as IL-2. This is due to the expression of the high affinity IL-2 receptor. This receptor causes this population to respond to the amount of IL-2, which is 100 times less than the effect it has on naive T cells. Thus, the use of this type of cell can detect the effects of very low doses of unknown growth factors that are not sufficient to induce proliferation in resting (naive) T cells.

The assay is performed as follows. PBMCs were isolated from human peripheral blood by F / H gradient centrifugation and 2 μg / ml PHA (Sigma, Sain).
t Louis, MO) in 10% FCS / RPMI for 3 days. The cells were then washed in PBS and 5 ng / ml human recombinant I
Culture for 3 days in 10% FCS / RPMI in the presence of L-2 (R & D Systems, Minneapolis, MN). The cells were washed and rested in starvation medium (1% FCS / RPMI) for 16 hours before the proliferation assay was started. Aliquots of cells are analyzed by FACS to determine the percentage of T cells (CD3 positive cells) present; this is usually 93-97% depending on the donor.
Spans between. The assay is performed in 96-well plates with 2 × 10 ⁴ cells / well in a final volume of 200 μl. Supernatants expressing the protein of interest (eg CHO or 293T supernatant) are tested at a final dilution of 30%, therefore 60 μl is added to 140 μl of 10% FCS / RPMI containing cells. Control supernatants are used at the same final dilution and express the following proteins: vector (negative control), IL-2, IFNγ, TNFα, IL-1.
0 and TR2. In addition to control supernatant, recombinant human IL-2 is also used at a final concentration of 10 ng / ml. After 24 hours of culture, each well is pulsed with 1 μCi of ³ H thymidine (Nen, Boston, MA). The cells are then pulsed 20
Harvested after time and ³ H thymidine incorporation is used as an indicator of proliferation. Results are expressed as the mean ± standard error of triplicate samples.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art will appreciate that the activity of the polynucleotides of the invention (eg, gene therapy)
, The exemplified studies can be readily modified to test agonists and / or antagonists of the polynucleotides or polypeptides of the invention.

Example 34: Effect of Polypeptides of the Invention on MHC Class II, Co-stimulatory and Adhesion Molecule Expression, and Cell Differentiation of Monocytes and Monocyte-Derived Human Dendritic Cells Produced by proliferation of proliferative progenitors found in blood: Adherent PBMC or cleared monocyte fractions were treated with GM-CSF (50 ng / ml) and I
Incubate with L-4 (20 ng / ml) for 7-10 days. These dendritic cells have characteristic phenotypes of immature cells (CD1, CD80, CD86, CD40 and M40).
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, downregulation of FCγRII, upregulation of CD83). Occurs. These changes are associated with increased antigen presenting capacity and functional maturation of dendritic cells.

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 to 3 days and 1% B
Wash with PBS containing SA and 0.02 mM sodium azide, then 1
: Incubate for 30 minutes at 4 ° C. with 20 dilutions of the appropriate FITC- or PE-labeled monoclonal antibody. After further washing, labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Effect on Cytokine Production Cytokines produced by dendritic cells, particularly IL-12, are important in the initiation of T cell dependent immune responses. IL-12
Strongly influence the development of a Th1 helper T cell immune response and induce cytotoxic T
Induces cellular and NK cell function. IL using ELISA as follows
-12 Release is measured. Dendritic cells (10 ⁶ / ml) are treated with increasing concentrations of a 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 (Minneap
olis, MN)) for analysis of IL-12 content. Use the standard protocol provided in the kit.

Effect on expression of MHC class II, costimulatory molecules and adhesion molecules. Three major families of cell surface antigens can be identified on monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptors. Modulation of the expression of MHC class II antigens and other costimulatory molecules such as B7 and ICAM-1 can result in changes in the ability of monocytes to present antigen and to induce T cell activation. Increased Fc receptor expression may be correlated with improved monocyte cytotoxic activity, cytokine release and phagocytosis.

FACS analysis is used to test surface antigens as follows. Monocytes were enriched at 1 with increasing concentrations of a polypeptide of the invention or LPS (positive control).
~ PB treated for 5 days and containing 1% BSA and 0.02 mM sodium azide
Wash with S, then incubate for 30 minutes at 4 ° C. with a 1:20 dilution of the appropriate FITC- or PE-labeled monoclonal antibody.
After further washing, labeled cells were washed with FACScan (Becton Dickin
Son) for analysis by flow cytometry.

Molecules that Increase Monocyte Activation and / or Survival Activate Monocytes (or Inactivate) and / or Increase Monocyte Survival (or Decrease Monocyte Survival) Assay is known in the art and can be routinely applied to determine whether the molecules of the invention function as monocyte inhibitors or activators. The polypeptides, agonists or antagonists of the invention can be screened using the three assays described below. For each of these assays, peripheral blood mononuclear cells (PBMC) were treated with Histop
Single donor leuko by centrifugation through an aque gradient (Sigma)
pack (American Red Cross, Baltimore, MD
). Countercurrent centrifugal elutriation (counterflo)
Isolate from PBMC by w centrifugal elution).

Monocyte Survival Assay Human peripheral blood monocytes progressively 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 (
PI) staining is used to measure apoptosis as follows. 100n for monocytes
Incubate for 48 hours in serum-free medium (positive control) in polypropylene tubes in the presence of g / ml TNF-α (negative control) and in the presence of various concentrations of the compound to be tested. Cells are suspended in PBS containing PI at a final concentration of 5 μg / ml to a concentration of 2 × 10 ⁶ / ml and then incubated for 5 minutes at room temperature before FACScan analysis. PI uptake has been shown to correlate with DNA fragmentation in this test paradigm.

Effect on Cytokine Release A key 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 to measure cytokine release is performed as follows.
Human monocytes are incubated at a density of 5 × 10 ⁵ cells / ml with increasing concentrations of the polypeptide of the invention and under the same conditions in the absence of this polypeptide. For production of IL-12, the cells are primed with IFN (100 U / ml) overnight in the presence of the polypeptides of the invention. Then, LPS (10
ng / ml) is added. Conditioned medium is collected after 24 hours and stored frozen until use. Then, the measurement of TNF-α, IL-10, MCP-1 and IL-8 is performed using a commercially available ELISA kit (for example, R & D Systems Minne.
apolis, MN) and applying the standard protocol provided in the kit.

(Oxidative burst) 96 purified monocytes.
Plate well plates at 2-1 × 10 ⁵ cells / well. Increasing concentrations of the polypeptides of the invention were added to wells in a total volume of 0.2 ml of culture medium (RPMI 1640+
10% FCS, glutamine and antibiotics). After 3 days of incubation, the plates are centrifuged and the medium is removed from the wells. In a monolayer of macrophages, 0.2 ml of phenol red solution (140 m
M NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U / ml HRPO),
Add with stimulant (200 nM PMA). This plate at 37 ℃ 2
Incubate for a period of time and stop the reaction by adding 20 μl of 1N NaOH per well. Read absorption at 610 nm. To calculate the amount of H ₂ O ₂ produced by macrophages, a standard curve of H ₂ O ₂ solution of known molarity is performed for each experiment.

The studies described in this example tested activity of a polypeptide of the invention. However, those skilled in the art will appreciate that the activity of the polypeptide of the invention, the activity of the polynucleotide (
For example, gene studies), agonist activity, and / or antagonist activity can be readily modified to study the illustrated studies.

Example 35 Biological Effects of Polypeptides of the Present Invention Astrocyte and Neural Assays [0946] As described above, recombinant polypeptides of the present invention expressed and purified in Escherichia coli were prepared. , The activity of promoting cortical neuronal cell survival, neurite outgrowth or phenotypic differentiation, and the induction of glial fibrillary acidic protein immunopositive cells, astrocyte proliferation. The selection of cortical cells for bioassays is based on the predominant expression of FGF-1 and FGF-2 in cortical structures and the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. Thymidine incorporation assays can be used, for example, to elucidate the activity of the polypeptides of the invention on these cells.

In addition, FGF to cortical or hippocampal neurons in vitro
A previous report describing the biological effects of -2 (basic FGF) demonstrated an increase in both neuronal survival and neurite outgrowth (Walicke et al., "Fibroblast growth factor promoters".
survival of dissociated hippocampal
neurons and enhancements neuroite extensi
on ”Proc. Natl. Acad. Sci. USA 83: 3012-30
16 (1986), the assay is hereby incorporated by reference in its entirety).
. However, reports from experiments carried out 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 on the target cells. Suggest that you may depend. The ability of the polypeptides of the invention to induce neurite outgrowth can be compared to the response obtained with FGF-2 using the primary cortical neuron culture paradigm, eg, using the thymidine incorporation assay.

Fibroblast and Endothelial Cell Assay Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.) And maintained in growth medium from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, CA). For proliferation assays, human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells / well in 96 well plate growth medium for 1 day. The cells are then incubated for 1 day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test protein for 3 days. Alamar Blue (Almar Bios
(Ciences, Sacramento, CA) is added to each well to a final concentration of 10%. The cells are incubated for 4 hours. Cell viability is C
Measure by reading with a ytoFluor fluorescence reader. For the PGE ₂ assay, human lung fibroblasts are cultured in 96-well plates for 1 day at 5,000 cells / well. After converting 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 was collected and the EIA kit (
Cayman, Ann Arbor, MI) by assaying for PGE _2. For the IL-6 assay, human lung fibroblasts are cultured in 96-well plates for 1 day at 5,000 cells / well. After converting the medium to 0.1% BSA basal medium, cells are incubated with FGF-2 for 24 hours with or without the polypeptide IL-1α of the invention. Supernatants were collected and IL by ELISA kit (Endogen, Cambridge, MA)
Assay for -6.

Human lung fibroblasts are cultured with FGF-2 or a polypeptide of the invention in basal medium for 3 days, then Alam to assess the effect on fibroblast proliferation.
Add ar Blue. FGF-2 should show a stimulus of 10 to 2500 ng / ml which could be used comparable to that with the polypeptides of the invention.

Parkinson's Model Loss of motor function in Parkinson's disease results from striatal dopamine deficiency resulting from degeneration of nigrostriatal dopaminergic projection neurons. A widely characterized animal model of Parkinson's disease is 1-methyl-4phenyl 1,2,3
, 6-tetrahydropyridine (MPTP) systemic administration. In the CNS,
MPTP is taken up by astrocytes and 1 by monoamine oxidase B
-Catabolized to methyl-4-phenylpyridine (MPP ⁺ ) and released.
Subsequently, MPP ⁺ actively accumulates in dopaminergic neurons by 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 interfering with electron transfer and To generate 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).
Et al., Dev. Biol. 1989). Recently, Unsicker's group has demonstrated that FGF-2 administration in striatal gel implants results in near complete protection of substantia nigra dopaminergic neurons from toxicity associated with MPTP exposure ( Otto and Unsicker, J. Neuroscience,
1990).

Based on the data using FGF-2, the polypeptide of the present invention exerts a similar effect to that of FGF-2 in enhancing the survival of dopaminergic neurons in vitro. Polypeptides of the invention can also be evaluated in vivo to determine if they have, and also tested in vivo for protection of dopaminergic neurons in the striatum from damage associated with MPTP treatment. The potential effects of the polypeptides of the invention are first tested in vitro in the dopaminergic neuronal cell culture paradigm. 14 days of pregnancy Wis
Cultures are prepared by dissecting the midbrain plate from the tar rat embryo. Tissues were dissociated with trypsin and plated on polyortinin-laminin coated coverslips at a density of 200,000 cells / cm ² . The cells are maintained in Dulbecco's modified Eagle medium and F12 medium containing hormone supplements (NI). After 8 days in vitro, cultures are fixed with paraformamide and processed for immunohistochemical staining with tyrosine hydroxylase, a specific marker for dopaminergic neurons. Separate cell cultures are prepared from embryonic rats. The culture medium is changed every 3 days and the factor is also added at each time point.

Since dopaminergic neurons are isolated from animals on day 14 of gestation (this time of development has passed the stage where dopaminergic progenitor cells proliferate), an increase in the number of tyrosine hydroxylase immunopositive neurons was observed in vitro. Shows an increase in the number of surviving dopaminergic neurons. Therefore, it is suggested that if the polypeptide of the present invention acts to prolong the survival of dopaminergic neurons, this polypeptide may be involved in Parkinson's disease.

The studies described in this example tested activity of a polypeptide of the invention. However, one 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 the activity of the agonists and / or antagonists of the invention.

Example 36 Effect of Polypeptides of the Invention on 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 at a density of 2-5 × 10 ⁴ cells per 35 mm dish in medium. On day 2, this medium was added to 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. Change medium on days 4 and 6. Day 8, Coulter C
Determine the cell number using an outer.

An increase in HUVEC cell number indicates that the polypeptides of the present invention are capable of proliferating vascular endothelial cells.

The studies described in this example tested activity of a polypeptide of the invention. However, one 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 the activity of the agonists and / or antagonists of the invention.

Example 37: Stimulatory Effect of Polypeptides of the Invention on Proliferation of Vascular Endothelial Cells A colorimetric assay using the electron-coupled reagent PMS (phenazine methosulfate) for evaluation of 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 serum-supplemented medium and allowed to attach overnight. 0
． After 12 hours serum starvation in 5% FBS, the wells (bFGF, VEGF ₁₆₅ or polypeptides 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 / PM
S 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 in an ELISA plate reader. Background absorbance of control wells (medium, no cells) is subtracted and 7 wells run in parallel for each condition. Leak et al., In Vitro Cell. Dev. Biol. 30A:
See 512-518 (1994).

The studies described in this example tested activity of a polypeptide of the invention. However, one 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 the activity of the agonists and / or antagonists of the invention.

Example 38 Inhibition of PDGF-Induced Vascular Smooth Muscle Cell Proliferation Stimulating Effect HAoSMC proliferation can be measured, for example, by BrdUrd incorporation. Briefly, subconfluent quiescent cells grown on 4-chamber slides were
Transfect with RP or FITC labeled AT2-3LP. The cells are then pulsed with 10% bovine serum and 6 mg / ml BrdUrd. 24
After hours, immunocytochemistry is performed by using the BrdUrd staining kit (Zymed Laboratories). Briefly, the cells are incubated with biotinylated mouse anti-BrdUrd antibody for 2 hours at 4 ° C. after exposure to denaturing solution, followed by streptavidin-peroxidase and diaminobenzidine. After counterstaining with hematoxylin, the cells are mounted for microscopic examination and Brd Urd-positive cells are counted. Brd
The Urd coefficient is calculated as the ratio of BrdUrd-positive cells to the total cell number. Furthermore, for individual cells, simultaneous detection of BrdUrd staining (nucleus) and FITC uptake (cytoplasmic) is performed by simultaneous use of bright and dark UV fluorescence illumination. (Hayashida et al., J. Biol. Chem. 6: 271 (
36): 211985-21992 (1996)).

The studies described in this example tested activity of a polypeptide of the invention.
However, one skilled in the art will appreciate that the activity of the polynucleotides of the invention (eg, gene therapy),
To test the activity of the agonists and / or antagonists of the invention,
The studies illustrated can easily be modified.

Example 39: Stimulation of Endothelial Migration This example can be used to explore the potential of the polypeptides of the invention to stimulate lymphoid endothelial cell migration.

The Endothelial Cell Migration Assay is Performed Using a 48-Well Microchemotactic Chamber (Neuroprobe Inc., Cabin John, MD; Falk).
W., J. 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) coated with 0.1% gelatin for at least 6 hours at room temperature,
Then it is dried under sterile air. Test substances are diluted to the appropriate concentration in M199 supplemented with 0.25% bovine serum albumin (BSA) and 25 μl of the final dilution is placed in the bottom chamber of the modified Boyden device. Subconfluent, early passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for the minimum time required for cell detachment. After placing the filter between the bottom chamber and the upper chamber, 2.5 × 10 ⁵ cells suspended in 50 μl M199 containing 1% FBS are seeded in the upper compartment. Then this device
Cells were allowed to migrate by incubating at 37 ° C. for 5 hours in a chamber moistened with 5% CO 2. After the incubation period, the filter was removed and the upper side of the filter with non-migrated cells was placed on the rubber 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 3 random high power fields (40x) of cells in each well. Then run all groups in quadruplicate.

The studies described in this example tested activity of a polypeptide of the invention.
However, one skilled in the art will appreciate that the activity of the polynucleotides of the invention (eg, gene therapy),
To test the activity of the agonists and / or antagonists of the invention,
The studies illustrated can easily be modified.

Example 40: Stimulation of nitric oxide production by endothelial cells The release of nitric oxide by vascular endothelium is believed to be a mediator of vascular endothelial relaxation. Thus, the activity of a polypeptide of the invention can be assayed by measuring nitric oxide production by endothelial cells in response to this polypeptide.

Nitric oxide was starved for 24 hours, followed by varying levels of positive control (
For example, in a 96-well plate of confluent microvascular endothelial cells after 4 hours of exposure to VEGF-1) and polypeptides of the invention. Nitric oxide in the medium is quantified by using the Griess reagent to measure the total amount of nitrite after reduction of nitrate derived from nitric oxide by nitrate reductase. The effect of the polypeptides of the invention on nitric oxide release is tested in HUVEC.

[0967] Briefly, NO release from cultured HUVEC monolayers was measured using a NO meter (Iso
-NO, World Precision Instruments Inc.
) (1049) connected to a NO-specific polarographic electrode.
The NO element is calibrated according to the following formula: 2KNO ₂ + 2KI + 2H ₂ SO ₄ 62NO + I ₂ + 2H ₂ O + 2K ₂ S
The O ₄ calibration curve was obtained by adding stepwise concentrations of KNO ₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol / L) in a calibration solution containing KI and H ₂ SO ₄ . obtain. The specificity of the Iso-NO electrode for NO is pre-determined by measuring NO from authentic NO gas (10
50). The culture medium is removed and the HUVECs are washed twice with Dulbecco phosphate buffered saline. The cells are then added to 5 wells in 6 well plates.
Immerse in ml of filtered Krebs-Henseleit solution, and place the cell plate in a slide warmer (Lab Li) to maintain the temperature at 37 ° C.
ne Instruments Inc. ) Hold. The NO sensor probe is inserted vertically into the well, keeping the tip of the electrode 2 mm below the surface of the solution before adding different conditions. S-nitrosoacetylpenicillamine (SNAP) is used as a positive control. The amount of NO released is expressed as picomolar concentration per 1 × 10 ⁶ endothelial cells. All reported values are for each group (number of cell culture wells)
Is an average of the measured values of 4 to 6. Leak et al., Biochem. and
Biophys. Res. Comm. 217: 96-105 (1995).

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonists, and / or antagonists of the polynucleotides of the invention.

Example 41 Effect of Polypeptides of the Invention on Cord Formation in Angiogenesis Another step in angiogenesis is 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.

CADMEC (microvascular endothelial cells) were used as proliferating cells (2 passages) in Cell
Applications Inc. Buy from Cell Applicat
Cultures in Ions' CADMEC Growth Medium and used at passage 5. For in vitro angiogenesis assay, wells of a 48-well cell culture plate were placed in Cell Applications'Attachment.
Use a Factor Medium (200 ml / well) at 37 ° C for 3
Coat for 0 minutes. CADMECs are seeded in wells coated with 7,500 cells / well and cultured overnight in Growth Medium. Then this Gr
owth Medium to control buffer or polypeptide of the invention (
300 mg Cell Applicati containing 0.1-100 ng / ml)
Replace with ons' Chord Formation Medium, and culture the cells for an additional 48 hours. The number and length of the capillary-like chords are calculated by Boeckel.
Quantification through the use of an ER VIA-170 video image analyzer. All assays were done in triplicate.

Commercially available (R & D) VEGF (50 ng / ml) is used as a positive control. b-estradiol (1 ng / ml) is used as a negative control. Appropriate buffer (no protein) is also used as a control.

The studies described in this example tested activity of a polypeptide of the invention. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonists, and / or antagonists of the polynucleotides of the invention.

Example 42: Angiogenic Effects on Chicken Chorioallantoic Membrane Chicken chorioallantoic membrane (CAM) is a well established system for testing angiogenesis. Angiogenesis in the CAM can be easily visualized and quantified. The ability of the polypeptides of the invention to stimulate angiogenesis in CAM can be tested.

Fermented eggs of Chinese walnut (Gallus gallus) and Japanese quail (Coturnix coturnix) were added to 37.8.
Incubate at 80 ° C and 80% humidity. Differentiated CA of 16-day-old chickens
M and 13 day old quail embryos are studied in the following manner.

On day 4 of development, a window is made in the eggshell of chicken eggs. The embryos are examined for normal development and the eggs are sealed with Cellotape®. These one more
Incubate until day 3. Thermanox cover glass (Nunc,
(Naperville, IL) are cut into discs of approximately 5 mm diameter. Sterile and salt-free growth factor is dissolved in sterile water, and about 3.3 mg / 5 ml is pipetted onto the disc. After air drying, the inverted disc is applied to the CAM. 3 days later, sample 3
Fixed in% glutaraldehyde and 2% formaldehyde, and 0.12
Rinse with M sodium cacodylate buffer. These are stereomicroscopes [Wild M8
] And photographed and embedded for semi-thin and ultra-thin sectioning as described above. Controls are performed with the carrier disc only.

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonists, and / or antagonists of the polynucleotides of the invention.

Example 43: Angiogenesis Assay Using Matrigel Implants in Mice [0977] An in vivo angiogenesis assay of the polypeptides of the present invention is based on the preexisting capillary network-like structure of mouse extracellular matrix material (Matrigel). ) To determine the ability to form new vessels in the implanted capsules. This protein is liquid Ma at 4 ° C.
It is mixed with trigel and then the mixture is injected subcutaneously into mice, where the mixture solidifies. After 7 days, Matrigel's solid "plugs" are removed and tested for the presence of new blood vessels. Matrigel, Bect
on Dickinson Labware / Collaborative B
Purchase from iomedical Products.

Matrigel material is a liquid when thawed at 4 ° C. This Matrix
1 is mixed with a polypeptide of the invention at 150 ng / ml at 4 ° C. and drawn into a cold 3 ml syringe. Female C57B1 / 6 mice, approximately 8 weeks old, are injected with a mixture of Matrigel and experimental protein at two sites on the midventral aspect of the abdomen (0
． 5 ml / site). After 7 days, the mice were sacrificed by cervical dislocation and Matrig
Remove the el plug and wash (ie, remove all attached membrane and fibrous tissue). Fix the same whole plug in neutral buffered 10% formamide,
After embedding in paraffin and staining with Masson's Trichrome, it is used to prepare sections for histological examination. Process cross sections from three different regions of each plug. Selected sections are stained for the presence of vWF. The positive control for this assay was bovine basic FGF (150n
g / ml). Matrigel alone is used to determine the basal level of angiogenesis.

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonists, and / or antagonists of the polynucleotides of the invention.

Example 44 Rescue of Ischemia in Rabbit Lower Extremity Model To study the in vivo effect of the polynucleotides and polypeptides of the invention on ischemia, a rabbit hindlimb ischemia model was previously described. (Takesh
ita et al., Am J. Pathol 147: 1649-1660 (1995).
), As 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 depends on collateral vessels arising from the internal iliac artery (Takeshita et al., Am J.
． Pathol 147: 1649-1660 (1995)). 10 day intervals allow post-surgical recovery of rabbits and development of endogenous collateral vessels. Surgery (0
Day) after 10 days, after performing a baseline angiography, the internal iliac artery of the ischemic limb was
Using 500 mg of naked expression plasmid containing the polynucleotide of the invention, (
Riessen et al., Hum Gene Ther. 4: 749-758 (199
3); Leclerc et al. Clin. Invest. 90: 936-944
Transfection by arterial gene transfer technique using a hydrogel-coated balloon catheter as described in (1992)). When the polypeptide of the invention is used for treatment, a single bolus of 500 mg of the polypeptide of the invention or control is delivered through the infusion catheter for 1 minute into the internal iliac artery of the ischemic limb. On day 30, various parameters are measured in these rabbits:
(A) BP ratio-ischemic limb systolic blood pressure to normal limb systolic blood pressure ratio; (b) blood flow and regurgitation-stop FL: blood flow during non-diastolic state and maximal FL: complete. Blood flow during the diastolic state, which is also an indirect measure of vascular volume, and regurgitation is maximal F
L: is reflected in the ratio of stopped FL; (c) Angiographic value (angiographic)
Score) -This is measured by angiography of the collateral vessels. Scores are determined by the percentage of circles in the overlaying grid (using transverse opacified arteries divided by the total number m of rabbit thighs); (d) capillary density-number of collateral capillaries. , Determined in light microscopy sections obtained from the hind limbs.

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 to test the agonists and / or antagonists of the invention.

(Example 45: Effect of the polypeptide of the present invention on vasodilation) Since dilation of vascular endothelium is important for lowering blood pressure, spontaneous hypertensive rats (S
The ability of the polynucleotides of the invention in HR) to influence blood pressure is tested. Increasing doses (0, 10, 30, 100, 300, and 900 mg / kg) of the polypeptides of the invention are administered to 13-14 week old spontaneously hypertensive rats (SHR). Data are expressed as mean +/- SEM. Statistical analysis is performed using a two-tailed t-test and statistical significance is defined as p <0.05 vs. response to buffer alone.

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonists, and / or antagonists of the polynucleotides of the invention.

Example 46: Rat ischemic skin flap model Evaluation parameters include skin blood flow, skin temperature, and factor VIII immunohistochemistry or endothelial alkaline phosphatase reaction. Expression of the polypeptides of the invention during skin ischemia is studied using in situ hybridization.

[0985]   The work in this model is divided into three parts:   a) Ischemic skin   b) Ischemic skin wound   c) Normal wound.

The experimental protocol includes: a) 3 × 4 cm single-stem full-thickness single pedicles.
A full-thickness random skin flap (a muscle flap over the lower back of the animal) is generated.

B) Make excisional wounds on ischemic skin (skin flap) (diameter 4-6 mm) c) Excisional wounds (0, 1, 2 after wounding) with the following various dosage ranges of the polypeptide of the invention. Local treatment on days 3, 4): 1 mg-100 mg d) for histological, immunohistochemical and in situ studies,
Wound tissue is collected 3, 5, 7, 10, 14, and 21 days after wounding.

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonists, and / or antagonists of the polynucleotides of the invention.

Example 47 Peripheral Artery Disease Model Angiogenic therapy with the polypeptides of the invention is a novel therapeutic strategy to obtain restoration of blood flow around ischemia in the case of peripheral arterial disease. . The experimental protocol includes: a) One unilateral femoral artery is ligated to create the hindlimb ischemic muscle, the other unilateral hindlimb serves as a control.

B) Polypeptides of the invention are delivered in a dosage range of 20 mg to 500 mg intravenously and / or intramuscularly three times per week (probably more) for 2-3 weeks.

C) This ischemic muscle tissue is collected at 1, 2, and 3 weeks after ligation of the femoral artery for analysis of the expression of the polypeptides of the invention and histology. Biopsies are also performed on the normal muscles of the other contralateral hind limb.

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonists, and / or antagonists of the polynucleotides of the invention.

Example 48: Ischemic Myocardial Disease Model The polypeptides of the invention are evaluated as potent mitogens that can stimulate collateral vessel development and reconstitute new blood vessels after coronary artery occlusion. . Altered expression of the polypeptide is investigated in situ. The experimental protocol includes: a) The heart is exposed via a left thoracotomy in the rat. Immediately, the left coronary artery,
Occlusate with a fine suture (6-0) and close the rib cage.

B) The polypeptides of the invention are delivered in a dosage range of 20 mg to 500 mg intravenously and / or intramuscularly three times per week (probably more) for 2 to 4 weeks.

C) Thirty days after surgery, the hearts are removed and cross-sectioned for morphometry and analyzed in situ.

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonists, and / or antagonists of the polynucleotides of the invention.

Example 49: Rat Corneal Wound Healing Model This animal model shows the effect of the polypeptides of the invention on neovascularization. The experimental protocol includes: a) making a long incision of 1-1.5 mm from the center of the cornea into the stromal layer b) inserting a spatula under the lip of the incision facing the outer edge of the eye c ) Make a pocket (its base is 1-1.5 mm from the eye edge) d) Place pellets containing 50 ng-5 μg (ug) of a polypeptide of the invention into the pocket e ) Treatment with the polypeptides of the invention may also be applied topically to corneal wounds within a dosage range of 20 mg to 500 mg (daily treatment for 5 days).

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity (eg, gene therapy), agonists, and / or antagonists of the polynucleotides of the invention.

Example 50: Diabetic Mice and Glucocorticoid Impaired Wound Healing Model A. Diabetic db + / db + Mouse Model To demonstrate that the polypeptides of the present invention promote the healing process, wound healing. The genetically diabetic mouse model of is used. The full thickness wound healing model in db + / db + mice is a well characterized, clinically relevant, and reproducible model of impaired wound healing. Healing of diabetic wounds depends on granulation tissue formation and re-epithelialization rather than contraction (Gartner, MH et al., J. Surg. Res. 52: 389 (
1992); Greenhalgh, D .; G. Et al., Am. J. Pathol. 1
36: 1235 (1990)).

This diabetic animal has many of the characteristic properties observed in type II diabetes mellitus. Homozygous (db + / db +) mice have their normal heterozygous (
db + / + m) obese compared to littermates. Mutant diabetes (db + / db +)
Mice carry a single autosomal recessive mutation on chromosome 4 (db +) (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, elevated 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 (N
orido, F.F. Et al., Exp. Neurol. 83 (2): 221-232 (1
984); Robertson et al., Diabetes 29 (1): 60-67.
(1980); Giacomelli et al., Lab Invest. 40 (4):
460-473 (1979); Coleman, D .; L. Diabetes 3
1 (Supplement): 1-6 (1982)). These homozygous diabetic mice develop hyperglycemia and are resistant to insulin, similar to human type II diabetes (Mandel et al., J. Immunol. 120: 1375-1377).
(1978)).

[1001] The features observed in these animals indicate that the healing in this model may be similar to that observed in human diabetes (Greenhalgh et al.,
Am. J. of Pathol. 136: 1235-1246 (1990)).

[1001] Genetically diabetic female C57BL / KsJ (db + / db +) mice and their non-diabetic (db + / + m) heterozygous littermates were used in this study (J
acksson Laboratories). These animals are purchased at 6 weeks of age. The study will start at 8 weeks of age. Animals are housed individually and given food and water ad libitum. All operations are performed using aseptic technique. This experiment, Human Ge
nome Sciences, Inc. Institutional Ani
mal Care and Use Committee and the G
uidelines for the Care and Use of La
Follow the Animal Health rules and guidelines.

[1003] The wound protocol was adapted from previously reported methods (Tsuboi, R. and Rif.
kin, D.M. B. J. 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 70% ethanol solution and iodine. The surgical area is dried with sterile gauze before wounding. Then, an 8 mm full-thickness wound
It is prepared using a Keyes tissue punch. Immediately after wounding, the surrounding skin is gently stretched to remove the spread of the wound. The wound is left open during the experiment. The application of treatment is topically applied for 5 consecutive days from the day of wounding. Prior to treatment, the wound is gently washed with sterile saline and gauze sponge.

[1004] The wounds are visually inspected and photographed at a fixed distance on the day of surgery and at 2 day intervals thereafter. Wound closure is determined by daily measurements on days 1-5 and 8. Calibrated wounds Jameson calipers (c
aliper) and measure horizontally and vertically. A wound is considered healed when granulation tissue is no longer visible and the wound is covered by continuous epithelium.

[1005] The polypeptides of the invention are administered in the vehicle for 8 days with different dosing ranges (4 mg to 500 mg per wound per day). The vehicle control group received 50 mL of vehicle solution.

[1006] 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.

[1007] Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) vehicle placebo control, 2) untreated group, and 3) treated group.

Wound closure is analyzed by measuring the area on the horizontal and vertical axes and obtaining the total area of the wound. Contraction is then assessed by establishing the difference between the area of the initial wound (day 0) and the area after treatment (day 8). This wound area on day 1 is 64 mm ² (size corresponding to the skin punch). Calculations are performed using the following formula: [Open area on day 8]-[Open area on day 1] / [Open area on day 1] Specimens 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 wounds is used to assess whether the healing process and morphological appearance of repaired skin have been altered by treatment with the polypeptides of the invention. This assessment includes verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization, and epidermal maturation (Green.
nhalgh, D.N. G. Et al., Am. J. Pathol. 136: 1235 (19
90)). Blinded lens micrometer with graduation
Observer).

[1009] 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 calibrated lens micrometer.

[1010] Proliferating cell nuclear antigen / cyclin (PCNA) in skin preparations was compared with ABC El.
Demonstrate by using anti-PCNA antibody (1:50) with the ite detection system. Human colon cancer can serve as a positive tissue control, and human brain tissue can be used as a negative tissue control. Each specimen contains sections with omission of primary antibody and replacement with non-immune mouse IgG. The rank of these sections is based on the extent of proliferation on a scale of 0-8 (lower scale reflecting slight proliferation to higher side reflecting vigorous proliferation).

[1011] Experimental data is analyzed using a one-tailed t-test. A p-value of <0.05 is considered significant.

B. Steroidopathic Rat Model Inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids an.
d Wound healing: Anti-Inflammatory St
eroid Action: Basic and Clinical Aspe
cts. 280-302 (1989); Wahl et al. Immunol. 1
15: 476-481 (1975); Werb et al., J. Am. 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., Growth Factors. 5: 295-304 (1991); Ha.
ynes et al. Clin. Invest. 61: 703-797 (1978).
), As well as causing a transient decrease in circulating monocytes (Ha
ynes et al. Clin. Invest. 61: 703-797 (1978).
Wahl, "Glucocorticoids and wound hair.
"Ling": Antiflammery Steroid Actio
n: Basic and Clinical Aspects, Academi
c Press, New York, 280-302 (1989)) to delay wound healing. Systemic administration of steroids for impaired wound healing is a well established phenomenon in rats (Beck et al., Growth Fac.
tors. 5: 295-304 (1991); Haynes et al. Clin
． Invest. 61: 703-797 (1978); Wahl, "Gluc.
occorticoids and wound healing ": Antii
nflammatory Steroid Action: Basic and
Clinical Aspects, Academic Press, New
York, 280-302 (1989); Pierce et al., Proc. Na
tl. Acad. Sci. USA 86: 2229-2233 (1989)).

[1013] To demonstrate that the polypeptides of the invention can promote the healing process, multiple topical applications of the polypeptide to rat full-thickness excised skin wounds, where healing is impaired by systemic administration of methylprednisolone. Evaluate the effect of.

Young adult male Sprague Dawley rats (weight 250-300 g)
) (Charles River Laboratories) is used for this experiment. The animals are purchased at 8 weeks of age. And the beginning of the study is 9 weeks of age. The healing response of the rat was determined by systemic administration of methylprednisolone (17 m
g / kg / rat, intramuscular). Animals are housed individually and given food and water ad libitum. All operations are performed using aseptic technique. This research
Institution of uman Genome Sciences, Inc.
al Animal Care and Use Committee and
the Guidelines for the Care and Use
Follow the rules and guidelines of of Laboratory Animals.

[1015] The wound protocol follows Section A above. On the day of wounding, animals are treated with ketamine (50
(mg / kg) and xylazine (5 mg / kg) intramuscularly. The dorsal area of the animal is shaved and the skin is washed with 70% ethanol and iodine solution. The surgical area is dried with sterile gauze before wounding. 8 mm full thickness wounds are created using a Keyes tissue punch. The left side of the wound is opened during the experiment. Application of the test substance is given topically once a day for 7 consecutive days, starting from the day of wounding and then methylmethylprednisolone administration. Prior to treatment, the wound is gently washed with sterile saline and gauze sponge.

[1016] 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 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.

[1017] The polypeptides of the present invention are administered in the vehicle for 8 days with different dose ranges (4 mg to 500 mg per wound per day). The vehicle control group received 50 mL of vehicle solution.

[1018] 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.

[1019] Four groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) untreated group, 2) vehicle placebo control, 3) treated group.

[1020] Wound closure is analyzed by measuring the area on the vertical and horizontal axes and obtaining the total area of the wound. Closure is then assessed by establishing the difference between the area of the initial wound (day 0) and the area after treatment (day 8). This wound area on day 1 is 64 mm ² (size corresponding to the skin punch). Calculations are performed using the following formula: [Open Area on Day 8]-[Open Area on Day 1] / [Open Area on Day 1] Specimens are fixed in 10% buffered formalin and paraffin. The embedded mass is cut perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H & E) staining is performed on transverse sections of bisected wounds. Histological examination of wounds 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 of the invention. A graduated lens observer is used by a blinded observer to determine the gap distance of the wound.

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 activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 51: Lymphedema Animal Model The purpose of this experimental approach is to test the therapeutic effect of the polypeptides of the invention on lymphangiogenesis and reestablishment of lymphatic circulatory system in rat hindlimbs. To create a suitable and consistent model of lymphedema. Efficacy is measured by swelling volume of the affected limb, quantification of lymphatic vascular mass, total plasma protein mass, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progression of edema lasts up to 3-4 weeks.

[1024] Prior to beginning surgery, blood samples are drawn for protein concentration analysis.
Male rats (weighing approximately 350 g) are dosed with pentobarbital. Then, the right limb is shaved from the knee to the hip joint. Wipe the shaved area with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. After measuring the circumference and volume, mark the two measurement levels (0.5 cm above the heel, midpoint of the back of the foot) and then inject the dye into the foot. On the endothelium of both right and left dorsal
Inject 0.05 ml of 1% Evan's Blue. Then, after the dye is injected into the paw, circumference measurement and volume measurement are performed.

[1025] Using the knee joint as a landmark, an incision in the groin of the middle limb is made in a ring so that the position of the femoral vessel can be confirmed. The flaps are dissected and separated using forceps and hemostat. After locating the femoral vessel, the lymphatic vessels that run alongside and below the vessel are located. The major lymphatic vessels in this area are then electrocoagulated suture ligated.

[1026] Using a microscope, the muscles of the back of the limb (semitendinosus)
And around the adductor muscle). The location of the popliteal lymph nodes is then confirmed. The two proximal lymphatic vessels and two distal lymphatic vessels as well as the distal blood supply of the popliteal node are ligated with sutures. The popliteal lymph nodes and any associated adipose tissue are then removed by cutting connective tissue.

[1027] Be careful to control any mild bleeding that occurs with this procedure. After occlusion of lymphatic vessels, flaps are placed on the liquid skin (Vetbond) (AJ Buck).
Seal by using. Separate skin edges are sealed to the underlying muscle tissue leaving a gap of about 0.5 cm around the leg. The skin may also be anchored by suturing to the underlying muscle when needed.

[1028] Animals are individually housed using mesh (no bedding) to avoid infection. Recovered animals are checked daily through the optimal edema peak. This peak typically occurs 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 the procedure 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 control and edematous limbs are evaluated at two points. The analysis is performed in a blind manner.

[1029] Circumference measurement: Under short-term gas anesthesia to prevent movement of the limb, the circumference of the limb is measured using a cloth tape measure. Measurements are made on the talar and dorsal paw by two different persons and then these two readings are averaged. Readings are taken from both control and edematous limbs.

[1030] Volumetric: On the day of surgery, animals are anesthetized with pentobarbital and tested prior to surgery. For daily volume measurements, animals are placed under short-term halothane anesthesia (rapid immobilization and quick recovery), both legs shaved, and the legs marked equally with water-resistant markers. The legs are first dipped in water, then dipped into the device to each marked level, then Buxco hydrops software (Chen / Victor
or)). The data is recorded by one person, while the other is soaked in the marked area of the leg.

Blood-plasma protein measurements: Blood is drawn prior to surgery, centrifuged, and serum separated, then the total protein and Ca2 + comparisons are concluded.

[1032] Limb Weight Comparison: After the blood is drawn, the animals are prepared for tissue collection.
The limb is cut with a quillitine, then both the experimental and control legs are ligated and cut and weighed. A second weighing is performed by removing the tibio-cakaneal joint and weighing the foot.

Histological Preparation: Transverse muscles located behind the knee (popliteal) area were excised and placed in metal molds, filled with freezeGel, soaked in cold methylbutane and -80EC until cut into labeled sample bags. Place it. Upon cutting, the muscle is observed under the fluorescent microscope for lymphatic vessels.

[1034] The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 52 Suppression of TNFα-Induced Adhesion Molecule Expression by Polypeptides of the Invention [1035] The recruitment of lymphocytes to areas of inflammation and angiogenesis is associated with cell surface adhesion molecules (CAMs) on lymphocytes. It relates to specific receptor-ligand interactions with the vascular endothelium. This adhesion process is associated with intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-) in both normal and pathological settings.
1), and following a multistep cascade involving endothelial leukocyte adhesion molecule-1 (E-selectin) expression in endothelial cells (EC). Expression of these and other molecules on the vascular endothelium determines the efficiency with which leukocytes can adhere to the local vasculature and overflow into local tissues during the development of the inflammatory response. Local concentrations of cytokines and growth factors are involved in regulating the expression of these CAMs.

[1036] Tumor necrosis factor alpha (TNF-alpha), a potent pro-inflammatory cytokine, is a stimulator of all three CAMs in endothelial cells and may be involved in a wide variety of inflammatory responses, often resulting in disease. Bring physical results.

[1037] The potential of the polypeptides of the invention to mediate the suppression of TNF-α induced CAM expression may be tested. A modified ELISA assay, which uses EC as the solid phase adsorbent, was used to quantify the amount of CAM expression in the TNF-α-processed protein EC when co-stimulated with members of the FGF family of proteins. taking measurement.

To perform this experiment, human umbilical vein endothelial cell (HUVEC) cultures were obtained from pooled cord harvests and 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 in 96 well plates at a concentration of 1 × 10 ⁴ cells / well in EGM medium at 37 ° C. for 18-24 hours or until confluent. The monolayers are subsequently washed 3 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 indicated cytokines and / or growth factors. Processed. After incubation, the cells are then evaluated for CAM expression.

[1039] 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 199 medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (10 μl volume). The plates are incubated at 37 ° C. for either 5 hours (selectin and integrin expression) or 24 hours (integrin expression only). Aspirate the plate to remove the medium and 100 μl of 0.1%
Paraformaldehyde-PBS (with Ca ++ and Mg ++) is added to each well. Hold the plate for 30 minutes at 4 ° C.

The fixative was then removed from the wells and the wells were PBS (+ Ca, Mg).
Wash once with + 0.5% BSA and drain. Do not dry this well. 10 μl of diluted primary antibody is added to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin
And anti-E-selectin-Biotin at a concentration of 10 μg / ml (0.1 mg / ml).
/ Ml stock antibody 1:10 dilution). The cells are incubated for 30 minutes at 37 ° C in a humidified environment. Wells with PBS (+ Ca, Mg
) Wash 3 times with + 0.5% BSA.

[1041] Then, 20 μl of diluted ExtrAvidin-Alkaline Ph
Add osphotase (1: 5,000 dilution) to each well and at 37 ° C.
Incubate for 30 minutes. Wells with PBS (+ Ca, Mg) + 0.5
Wash 3 times with% BSA. 1 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 transferred to ExtrAvidin-Alkaline Pho in glycine buffer.
Working dilution of spotase (1: 5
000 (10 ⁰ )> 10 ^−0.5 > 10 ⁻¹ > 10 ^−1.5 ). 5
μl of each dilution was added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNPP reagent must then be added to each standard well. The plate must be incubated at 37 ° C for 4 hours. Three
A volume of 50 μl of M NaOH is added to all wells. The results are quantified on a plate reader at 405 nm. The background subtraction option is used on 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]. Results are shown as the amount of bound AP conjugate in each sample.

The studies described in this example tested activity of a polypeptide of the invention.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of the polynucleotides (eg, gene therapy), agonists, and / or antagonists of the invention.

Example 53: Assay for Stimulation of Bone Marrow CD43 + Cell Proliferation This assay is based on the ability of human CD34 + to proliferate in the presence of hematopoietic growth factor and this assay is expressed in mammalian cells. The isolated polypeptide is evaluated for its ability to stimulate proliferation of CD34 + cells.

[1044] The most mature precursors have previously been shown to respond to a single signal only. The more immature precursors require at least two signals to respond. Thus, in order to test the effect of a polypeptide on hematopoietic activity of a wide range of progenitor cells, this assay was performed in the presence or absence of other hematopoietic growth factors.
Contains a given polypeptide. Combining the isolated cells with a test sample,
Culture for 5 days in the presence of stem cell factor (SCF). Only SCF has bone marrow (BM
) It has a very limited effect on the growth of cells and acts only under these conditions as a "survival" factor. However, when combined with any factor that exhibits a stimulatory effect on these cells (eg IL-3), SCF produces a synergistic effect. Thus, if the tested polypeptide has a stimulatory effect on hematopoietic ancestry, such activity can be readily detected. Normal BM cells have a low level of cell cycle, so it appears that no inhibitory effect of the desired polypeptide, or its agonists or antagonists, is detected. Therefore, assays for inhibitory effects on ancestors preferably
Tested in cells that are first subjected to in vitro stimulation with CF + IL + 3 and then contacted with the compound being evaluated for inhibition of such induced proliferation.

[1045] Briefly, CD34 + cells are isolated using methods known in the art.
These cells were thawed and cultured in medium (QB with 1% L-glutamine.
SF 60 Serum-free medium (500 ml) (Quality Biologi)
cal Inc. , Gaithersburg, MD Cat # 160-20.
4-101)). After some gentle centrifugation at 200 xg, the cells are left for 1 hour. Adjust the cell number to 2.5 × 10 ⁵ cells / ml.
During this time, 100 μl of sterile water is added to the peripheral wells of the 96 well plate. Cytokines that can be tested for a given polypeptide in this assay include 50 ng / ml rhSCF (R & D Systems, Minneapol.
is, MN, Cat # 255-SC) and 30 ng / ml r.
hSCF and rhIL-3 (R & D Systems, Minneapolis,
MN, Cat # 203-ML). After 1 hour, 10 μl of the prepared cytokine, 50 μl of supernatant (supernatant at 1: 2 dilution = 50 μl) and 20 μl of diluted cells were added to the medium, which medium had a final total volume of 100 μl. Already in the well to. The plate is then 37
Place in C / 5% CO ₂ incubator for 5 days.

[1064] 18 h prior to assay, 0.5 μCi / well [[
3H] thymidine is added to each well in a volume of 10 μl and the growth rate is determined. This experiment used Tomtec Harvester 96 to load cells 9 times each.
Termination is done by collecting from 6-well plates into filter mats. After collection, the filter mats are dried, trimmed and placed in one OmniFilte.
OmniFilter consisting of r-plate and one OmniFilter tray
er assembly. Add 60 μl Microscint to each well,
The plate is then sealed with TopSeal-A press-on film. Secure the barcode 15 sticker to the first plate for counting. The sealed plate was then filled and the level of radioactivity adjusted to Packard Top C
Data is collected for analysis, determined through the printed and printed. Radioactivity level reflects the amount of cell proliferation.

The studies described in this example test the activity of certain polypeptides that stimulate bone marrow CD34 + cell proliferation. One of ordinary skill in the art can readily modify the illustrated examples to test the activity of polynucleotides (eg, gene therapy), antibodies, agonists and / or antagonists, and fragments and variants thereof. As a non-limiting example, antagonists that may be tested in this assay are those that inhibit cell proliferation in the presence of cytokines and / or inhibit cell proliferation in the presence of cytokines and certain polypeptides. Is expected to increase. In contrast, agonists that may be tested in this assay are expected to enhance cell proliferation and / or reduce inhibition of cell proliferation in the presence of cytokines and certain polypeptides.

The ability of the gene to stimulate proliferation of bone marrow CD34 + cells indicates that the polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and treatment of disorders affecting the immune system and hematopoiesis. Typical uses are described in the "Immune Reactivity" and "Infectious Diseases" sections above, and elsewhere herein.

Example 54: Assay for Extracellular Matrix Enhanced Cellular Response (EMECR) The purpose of the Extracellular Matrix Enhanced Cellular Response (EMECR) assay is to identify hematopoietic stem cells in relation to extracellular matrix (ECM) induced signals. To identify gene products that act on (eg, isolated polypeptides).

[1058] In response to signals received from the surrounding microenvironment, cells respond to regulatory factors. For example, fibroblasts, and endothelial and epithelial stem cells are unable to replicate in the absence of signal from the ECM. Hematopoietic stem cells can undergo self-renewal in bone marrow, but not in in vitro suspension culture. The ability of stem cells to undergo self-renewal in vitro depends on their interaction with stromal cells and the ECM protein fibronectin (fn). Cell f
adsorption to n is, α _5. β ₁ and α ₄ . Mediated by β ₁ integrin receptors, these receptors are expressed by human and mouse hematopoietic stem cells. The factors responsible for integrating in the ECM environment and stimulating self-renewal of stem cells have not yet been identified. The discovery of such factors is an important goal in gene therapy and spinal cord transplant applications.

[1051]   Briefly, 96-well plates without tissue culture treatment of polystyrene are
0.2 μg / cm^TwoCoated with fn fragment at a coating concentration of. Ma
Mouse bone marrow cells were plated in 0.2 ml serum-free medium (1,000 cells / well).
Rate In the presence of IL-3 (5 ng / ml) + SCF (50 ng / ml)
Cultured cells are not expected to undergo stem cell self-renewal, and are expected to undergo distinct differentiation.
In some cases, it acts as a positive control. The gene product is SCF (5.0
ng / ml) with or without a suitable negative control.
, Where the test agent supernatant represents 10% of the total assay volume. Next
Plated cells in a hypoxic environment (5% CO_Two, 7% O_TwoAnd 88% N _Two ) Was grown by incubating in a tissue culture incubator for 7 days.
Let The number of proliferating cells in the wells was then measured for thymidine incorporation into cellular DNA.
Quantify by setting. Confirmation of positive hits in the assay is
Cell phenotypic characterization, which scales up the culture system.
, And using appropriate antibody reagents for cell surface antigens and FACScan
To be achieved.

[1052] One of skill in the art can readily modify exemplary studies to test the activity of polynucleotides (eg, gene therapy), antibodies, agonists and / or antagonists and fragments and variants thereof.

[1053] If a particular gene product is found to be a stimulator of hematopoietic ancestry, then polynucleotides and polypeptides corresponding to this gene may be useful in diagnosis and in the treatment of disorders that affect the immune system and hematopoiesis. possible. Typical applications are the above "
It is described in the section "Immune Activity" and "Infectious Diseases", as well as elsewhere herein. Gene products are also found in various blood chain stem cells and precursor cells (commites).
d progenitor) and in the differentiation and / or proliferation of various cell types.

[1054] Additionally, polynucleotides and / or polypeptides of the gene of interest, and / or agonists and / or antagonists thereof, may also be used to inhibit hematopoietic cell proliferation and differentiation, and thereby induce chemotherapy. In the meantime, it can be used to protect bone marrow stem cells from chemotherapeutic agents. This anti-proliferative effect may allow for the administration of higher doses of chemotherapeutic agents and thus more effective chemotherapeutic treatment.

[1055] In addition, polynucleotides and polypeptides corresponding to the gene of interest may also be associated with hematopoietic-related disorders (eg, anemia, pancytopenia, leukopenia, thrombocytopenia,
Or leukemia) may be useful in the treatment and diagnosis. This is because stromal cells are important in the generation of hematopoietic lineage cells. These uses include bone marrow cell ex vivo culture, bone marrow transplantation, bone marrow repopulation, neoplastic radiotherapy or chemotherapy.

Example 55: Proliferation of Human Dermal Fibroblasts and Aortic Smooth Muscle Cells The polypeptide of interest was added to a culture of normal human dermal fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC). Add and perform two simultaneous assays with each sample. The first assay is for normal human fibroblasts (NH
The effect of the polypeptide of interest on the proliferation of DF) or aortic smooth muscle cells (AoSMC) is tested. Abnormal proliferation of fibroblasts or smooth muscle cells is part of several pathological processes, including fibrosis and restenosis. The second assay tests IL6 production by both NHDF and SMC. IL6 production is an indicator of functional activation, activated cells have an increase in the production of numerous cytokines and other factors, which is proinflammatory.
) Or may have immunomodulatory consequences. The assay is performed with co-TNFa stimulation to check co-stimulatory or inhibitory activity, and co-TNFa
Do without stimulation.

[1057] Briefly, on day 1, 96-well black plates were plated at 1000 cells / well (NHDF) or 2000 cells / well (AoS) in 100 μl culture medium.
MC). NHDF culture medium contains the following: Clonetics FB basal medium, 1 mg / ml hFGF, 5 mg / ml insulin, 50 mg / ml gentamicin, 2% FBS, while AoSMC culture medium is Cloneti.
cs SM basal medium, 0.5 μg / ml hEGF, 5 mg / ml insulin,
1 μg / ml hFGF, 50 mg / ml gentamicin, 50 μg / ml A
mphotericin B, containing 5% FBS. At least 4-5 at 37 ° C
After incubation for a period of time, the culture medium is aspirated and replaced with growth arrest medium. N
Growth arrest medium for HFD comprises fibroblast basal medium, 50 mg / ml gentamicin, 2% FBS, while growth arrest medium for ApSMC is SM basal medium,
50 mg / ml gentamicin, 50 μg / ml Amphotericin
B, containing 0.4% FBS. Incubate at 37 ° C until day 2.

[1058] On day 2, serial dilutions of the polypeptide of interest and template are designed such that they always contain a media control and a known protein control. For both stimulation and inhibition experiments, proteins are diluted in growth arrest medium, for inhibition experiments TNFa is added to a final concentration of 2 ng / ml (NHDF) or 5 ng / ml (AoSMC). Control or 1/3 volume of medium containing the polypeptide of the invention was added and until day 5 37 ° C / 5%
Incubate in CO ₂ .

[1058] Transfer 60 μl from each well to another labeled 96-well plate, cover with plate sealer, and store at 4 ° C. (for IL6 ELISA) until day 6.
The remaining 100 μl in the cell culture plate is aseptically added with an amount equal to 10% of the culture volume (10 μl) of Alamar Blue. Plate 3-4
Return to incubator for time. Excitation at 530 nm and 590n
Fluorescence with emission at m is measured using a CytoFluor. This yields growth stimulation / inhibition data.

[1060] On day 5, IL-6 ELISA was diluted 50-1 with PBS (pH 7.4).
Performed by coating 96 well plates with 00 μl / well of anti-human IL-6 monoclonal antibody and incubating ON at room temperature.

[1061] On day 6, empty the plate contents into a sink and blot on a paper towel.
Prepare assay buffer with PBS containing 4% BSA. Plate the PB
Block with 200 μl / well of Pierce Super Block blocking buffer in S for 1-2 hours, then wash buffer (PBS, 0.05%).
Wash plate with Tween-20). Aspirate the plate on a paper towel. Then, 50 μl / well of anti-human IL-6 monoclonal biotin-labeled antibody diluted to 0.50 mg / ml is added. Make dilutions of IL-6 stock in medium (30 ng / ml, 10 ng / ml, 3 ng / ml, 1 ng / ml
, 0.3 ng / ml, 0 ng / ml). Duplicate samples are added to the top row of the plate. Cover plate and incubate on shaker for 2 hours at room temperature.

[1062] Plates are washed with wash buffer and blotted on paper towels. EU labeled streptavidin was diluted 1: 1000 in assay buffer and 100 μl /
Add wells. Cover plate and incubate at room temperature for 1 hour.
Wash plate with wash buffer. Blot on a paper towel.

Add 100 μl / well of Enhancement Solution and shake for 5 minutes. Walla
c Read plate on DELFIA Fluorometer. The readings from triplicate samples in each assay are tabulated and averaged.

[1064] A positive result in this assay is indicative of AoSMC cell proliferation, and that the gene product of interest may be involved in skin fibroblast proliferation and / or smooth muscle cell proliferation. A positive result also indicates a polypeptide, polynucleotide, polynucleotide / polypeptide agonist of the gene / gene product of interest and / or
Or suggests many potential uses for antagonists. For example, inflammation and immune responses, wound healing, and angiogenesis, as described throughout the specification. In particular, the polypeptide of this gene product and the polynucleotide of this gene can be used for wound healing and skin regeneration, and for promoting angiogenesis (both blood vessels and lymph vessels). Vascular growth can be used, for example, in the treatment of cardiovascular disease. Further, the antagonist of the polypeptide of the gene product and the polynucleotide of the gene acts by acting as anti-angiogenic (eg, anti-angiogenic) to cause diseases, disorders, and / or disorders involving angiogenesis. It may be useful in treating the condition. These diseases, disorders, and / or conditions,
For example: malignant tumors, solid tumors, benign tumors (eg, hemangiomas, acoustic neuromas, neurofibromas, trachoma, and pyogenic granulomas); artherosclerotic plaques.
cleric plaque); ocular angiogenic disorders (eg, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal transplant rejection, neovascular glaucoma, post lens fibroplasia, flushed skin, retinoblastoma, grape) Membranitis, and Pterygium of the eye (abnormal blood vessel growth)); Rheumatoid arthritis; Psoriasis; Delayed wound healing; Endometriosis; Angiogenesis; Granulation; Hyperplastic scars (keloids); Nonunion fracture
scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-We
bber syndrome; plaque neovascularization; telangiectasia; hemophilic joints; angiofibromas; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis, etc. in the art. Known and / or described herein. Moreover, antagonists of the polypeptide of the gene product and polynucleotide of the gene treat anti-hyperproliferative and / or anti-inflammatory diseases known in the art and / or described herein. Can be useful in doing so.

[1065] One of skill in the art can readily modify the illustrated studies to test the activity of polynucleotides (eg, gene therapy), antibodies, agonists and / or antagonists, and fragments and variants thereof.

Example 56: Expression of Cell Adhesion Molecules (CAM) on Endothelial Cells [1067] The recruitment of lymphocytes to the areas of inflammation and angiogenesis is associated with cell surface adhesion molecules (CAM) on lymphocytes. Includes specific receptor-ligand interactions with the vascular endothelium. The adhesion process follows a multi-step cascade in both normal and pathological environments, which involves intracellular adhesion molecule 1 (ICAM-1), vascular cell adhesion on endothelial cells (EC). Includes expression of molecule 1 (VCAM-1), and endothelial leukocyte adhesion molecule 1 (E-selectin). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes can adhere to local vasculature and overflow into local tissues during the progression of the inflammatory response. Local concentrations of cytokines and growth factors are involved in regulating the expression of these CAMs.

[1067] Briefly, endothelial cells (eg, human umbilical vein endothelial cells (HUVEC)) grow to confluence in standard 96-well plates. Growth medium was removed from the cells and 100 μl of 199 Medium (10% fetal bovine serum (FBS
)) Samples for testing and positive or negative controls are added to triplicate plates (10 μl volume). The plates are then incubated at 37 ° C for either 5 hours (selectin and integrin expression) or 24 hours (integrin expression only). The plate is aspirated to remove the medium and 100 μl of 0.1% paraformaldehyde-PBS (containing Ca ++ and Mg ++) is added to each well. Hold the plate at 4 ° C. for 30 minutes. The fixative was removed from the wells and the wells were washed with PBS.
Wash once with (+ Ca, Mg) + 0.5% BSA and drain. 10 μl
Of diluted primary antibody is added to the test and control wells. Anti-I
CAM-1-biotin, anti-VCAM-1-biotin, and anti-E-selectin-
Biotin is used at a concentration of 10 μg / ml (1:10 dilution of 0.1 mg / ml stock antibody). The cells are incubated at 37 ° C for 30 minutes in a humidified environment. Wells are washed 3 times with PBS (+ Ca, Mg) + 0.5% BSA. 20 μl of diluted ExtrAvidin-Alkaline Phosp
hotase (1: 5,000 dilution, referred to as a practical diluent in this specification)
Is added to each well and incubated for 30 minutes at 37 ° C. The well
Wash 3 times with PBS (+ Ca, Mg) + 0.5% BSA. One tablet of p-nitrophenol phosphate (pNP) per 5 ml of glycine buffer (pH 10.4)
Dissolve P). Add 100 μl pNPP substrate in glycine buffer to each test well. Three-part standard wells were filled with ExtrAvidi in glycine buffer.
n-Alkaline Phosphotase; 1: 5,000 (10 0)>
Prepare from a practical diluent of 10 ^−0.5 > 10 ⁻¹ > 10 ^−1.5 . When 5 μl of each dilution is added to a tripartite well, the resulting AP content in each well is 5.5 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent is then added to each standard well. This plate
Incubate at 37 ° C for 4 hours. A volume of 50 μl of 3M NaOH is added to all wells. The plate is read on a 405 nm plate reader using the background removal option with blank wells filled with glycine buffer only. In addition, template was added to each standard well [5.50 ng;
74 ng; 0.55 ng; 0.18 ng]. Results are shown as the amount of bound AP copolymer in each sample.

Example 57: Alamar Blue Endothelial Cell Proliferation Assay Using this assay, bFGF induction of bovine lymphatic endothelial cells (LEC), bovine aortic endothelial cells (BAEC), or human microvascular uterine myocytes (UTMEC). Protein-mediated inhibition of growth can be quantitatively determined. This assay incorporates a fluorometric growth indicator based on the detection of metabolic activity. Standard Alam
10 ng with ar Blue proliferation assay added as a source of endothelial cell stimulation
Condition in EGM-2MV containing 1 / ml bFGF. This assay can be used with a variety of endothelial cells with slightly changed growth media and cell concentrations. Appropriately dilute the dilution of the protein batch to be tested. Serum-free medium without bFGF (BIBCO SFM) was used as an unstimulated control, and Angiostatin or TSP-1 was included as a known inhibition control.

[1069] Briefly, LEC, BAEC or UTMEC were added to 96-well plates in 5 wells.
Seed the growth medium at a density of 000-2000 cells / well and leave at 37 ° C. overnight. After overnight incubation of the cells, growth medium is removed and replaced with GIBCO EC-SFM. The cells are treated with the appropriate dilutions of the protein of interest or control protein samples (prepared in SFM) in 3 wells containing additional bFGF to a concentration of 10 ng / ml. Once the cells are treated with the sample, the plate (s) is returned to the 37 ° C. incubator for 3 days. After 3 days, 10 ml of stock Alamar Bl
ue (Biosource Cat # DAL1100) is added to each well and the plate (s) is returned to the 37 ° C. incubator for 4 hours. The plate (s) is then read using a CytoFluor fluorescence reader with 530 nm excitation and 590 nm emission. Direct output is recorded in relative fluorescence units.

[1070] Alamar Blue is a redox indicator that fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth. As the cells grow in the medium, the innate metabolic activity causes a chemical reduction of the immediate environment.
Proliferative reduction can change the indicator from the oxidised (non-fluorescent blue) form to the reduced (fluorescent red) form. That is, stimulated proliferation produces a stronger signal, and inhibited proliferation produces a weaker signal, and the total signal is proportional to the total number of cells as well as their metabolic activity. Background levels of activity are observed with starvation medium only. This is compared to the output observed from the positive control sample (bFGF in growth medium) and protein dilution.

Example 58 Detection of Inhibition of Mixed Lymphocyte Response This assay is used to detect and evaluate inhibition of mixed lymphocyte reaction (MLR) by gene products (eg, isolated polypeptides). You can Inhibition of MLR may be due to direct effects on cell proliferation and viability, modulation of costimulatory molecules in interacting cells, modulation of adhesion between lymphocytes and accessory cells, or modulation of cytokine production by accessory cells. . Multiple cells can be targeted by these polypeptides. Because the peripheral blood mononuclear fraction used in this assay is
This is because it contains T, B and natural killer lymphocytes, as well as monocytes and dendritic cells.

[1072] The polypeptides of interest found to inhibit MLR may find use in diseases involving lymphocyte and monocyte activation or proliferation. This includes asthma, arthritis, diabetes, inflammatory skin conditions, psoriasis, eczema, systemic lupus erythematosus,
Such as multiple sclerosis, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, arteriosclerosis, cirrhosis, graft versus host disease, graft versus host disease, hepatitis, leukemia and lymphoma Diseases include, but are not limited to.

[1073]   Briefly, PBMCs derived from human donors were transferred to Lymphocyte Separ.
ation Medium (LSM (registered trademark), density 1.0070 g / ml
, Organon Teknika Corporation, West Ch
purification by density gradient centrifugation using Ester, PA). Two donna
-Derived PBMC supplemented with 10% FCS and 2 mM glutamine RPM
I-1640 (Life Technologies, Grand Island)
2 x 10 in d, NY)⁶Adjust to cells / ml. PBMC from a third donor
2 x 10⁵Adjust to cells / ml. 50 μl PBMC from each donor
Add to wells of 96 well round bottom microtiter plate. Dilution of test material
Aliquots (50 μl) are added to microtiter wells in triplicate. (Target tamper
Test sample 1: 4) to final dilution of 1: 4; rhuIL-2 (R
& D Systems, Minneapolis, MN, Catalog No. 202
-IL) was added to a final concentration of 1 μg / ml; anti-CD4 mAb (
R & D Systems, Clone 34930.11, Catalog Number MAB
379) is added to a final concentration of 10 μg / ml. Cells with 5% CO _Two Incubate for 7-8 hours at 37 ° C. in medium and 1 μC [[^Three[H] thymidine
Add to wells in last 16 hours. Collect cells and uptake thymidine
, Packard TopCount. Data is measured in three
Expressed as mean and standard deviation of constant values.

[1068] Samples of the protein of interest are screened in separate experiments and treated with a negative control treatment, anti-DC4 mAB (which inhibits lymphocyte proliferation), and a positive control treatment, IL-2 (recombinant material). Or as a supernatant) (this is
Promotes the proliferation of lymphocytes).

[1075] One of skill in the art can readily modify the exemplary studies to test the activity of polynucleotides (eg, gene therapy), antibodies, agonists and / or antagonists, and fragments and variants thereof.

It will be apparent that the present invention may be practiced other than as specifically described in the foregoing description and examples. Many modifications and variations of the present invention are possible in light of the above teachings, and are therefore within the scope of the appended claims.

[1077] Full disclosure of each document cited in the Background, Detailed Description and Examples (including patents, patent applications, academic literature, abstracts, laboratory manuals, books or other disclosures).
Are incorporated herein by reference. Further, both the hard copy of the Sequence Listing and the corresponding computer readout form, submitted with this specification, are hereby incorporated by reference in their entirety. Furthermore, US provisional application No. 60 /
Nos. 162,238 and 60 / 215,134 are incorporated herein by reference in their entirety.

[1078]

[Table 11] ATCC Deposit No. PTA-845 page 2 (Canada) Applicant is the Commissioner of the Patent Office until a Canadian patent is issued based on the application or the application is refused or abandoned and cannot be recovered or withdrawn. (Commissioner of Patents) requested that only the independent expert nominated by the Commissioner grant a sample of the deposited biological material referred to in the application, and the applicant Before the regulatory preparations for the publication of an application are completed, the International Bureau must be notified in writing.

[1077] (Norway) Applicants hereby agree that the application of samples is not subject to specialization in the art 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. Claim that it will only be done to the house. A request to this effect shall be made by the applicant to the Norwegian Patent Office before the time when the application is made publicly available under section 22 and section 33 (3) of the Norwegian Patent Act. If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts prepared by the Norwegian Patent Office (list of
any of those listed in "recognized experts", or
It can be any person approved by the applicant in the individual case.

[1080] (Australia) The Applicant hereby states that the donation of a sample of microorganisms is prior to the grant of the patent
Alternatively, before the abandonment, rejection or withdrawal of the application, a person who is a person skilled in the art who has no interest in the invention (skilled addre)
It is to be announced that it will only be performed for Ssee) (Australian Patent Law No. 3.25 (3)).

[1081] (Finland) The applicant hereby states that the application (Patent and Control Committee (National
That samples will only be provided to experts in the art until published (by Board of Patents and Regulations) or until a decision by the National Patent and Legal Commission is made without publication. ,Claim.

[1082] (UK) Applicants hereby claim that microbial samples are made available only to professionals. A request to this effect must be made by the applicant to the International Bureau before the regulatory preparations for the international publication of the application are complete. ATCC Deposit No. PTA-845 page 3 (Denmark) Applicants hereby provide samples until the application has been published (by the Danish Patent Office) or has not been published and has been decided by the Danish Patent Office. Claims that this is only done to experts in the art. A request to this effect shall be filed by the applicant with the Danish Patent Office before the time when the application is made publicly available under Articles 22 and 33 (3) of the Danish Patent Act. If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts prepared by the Danish Patent Office (list of).
any of those listed in "recognized experts", or
It can be any person approved by the applicant in the individual case.

[1083] (Sweden) The applicant hereby considers that the provision of samples shall be the subject matter of the art until the application has been published (by the Swedish Patent Office) or has been decided by the Swedish Patent Office without publication. Claim that it will only be done to the house. A request to this effect shall be filed by the applicant with the International Bureau not later than 16 months from the priority date (preferably PCT Applicant's Gui).
Form PCT / RO / 13 described in Annex Z of Volume I of de
4). If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts (lis) prepared by the Swedish Patent Office.
any of the persons listed in to of recognized experts),
Alternatively, it may be any person approved by the applicant in the individual case.

[1084] (Netherlands) Applicants are required to specialize in microorganisms based on the provisions of Patent Act 31F (1) until the date of issue of a Dutch patent, or until the date of rejection, withdrawal or lapped of an application. Claim that it will only be done in the form of sample delivery to the house. A request for this shall be made by the applicant before the Dutch industrial property right before the date on which the application is made publicly available under Article 22C or Article 25 of the Dutch Patent Act, whichever is earlier. It shall be submitted to the Bureau.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 1/04 A61P 7/00 4C084 3/10 7/02 4C085 5/00 7/04 4C087 7/00 7 / 06 4H045 7/02 9/00 7/04 9/10 7/06 11/06 9/00 13/12 9/10 21/04 11/06 25/00 13/12 25/16 21/04 25 / 28 25/00 29/00 25/16 101 25/28 31/12 29/00 31/18 101 33/00 31/12 35/00 31/18 35/02 33/00 37/02 35/00 37 / 08 35/02 C07K 14/47 37/02 16/18 37/08 C12N 1/15 C07K 14/47 1/19 16/18 1/21 C12N 1/15 C12P 21/02 C 1/19 C12Q 1/02 1/21 1/68 A 5/10 G01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/02 33/53 D 1/68 A61K 35/76 G01N 33/15 39/395 D 33/50 N 33/53 C12N 15/00 ZNAA // A61K 35/76 5/00 A 39/395 A61 37/02 37/24 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD) , SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB , BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT , TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Ruben, Stephen M. United States Maryland 20832, Ornay, Heritage Hills Drive 18525 (72) Inventor Comat Souris, George A. United States Maryland 20901, Silver Spring, Garwood Street 9518 (72) Inventor Duane, Roxanne Dee. United States Maryland 20817, Bethesda, Northfield Road 5515 (72) Inventor Fisella, Michael United States Maryland 20817, Bethesda, Red Wing Road 6308 (72) Inventor Eppner, Reinhardt United States Maryland 20878, Gaithersburg, number 316, Shelburne terrace 9906 F-term (reference) 2G045 AA40 DA36 FB03 4B024 AA01 AA11 BA53 BA80 CA04 DA02 DA06 EA02 EA04 FA18 GA03 GA11 HA01 HA12 HA17 4B063 QA01 QA18 QA19 QQ02 QQ03 QQ08 QQ13 QQ42 QQ53 QQ79 QR56 QR62 QR77 QR80 QS12 QS24 QS38 4B064 AC01 AG27 CA02 CA10 CA19 CA20 CC24 DA01 DA13 4B065 AA26X AA90X AA92X AA93Y AB01 AB05 AC14 BA02 BA08 CA24 CA25 CA44 CA46 4C084 AA02 AZA07 AA13 BA17 BA01 BA10 ZA512 ZA 522 ZA 522 ZA 522 ZA 152 ZA 522 ZA 152 2 ZB072 ZB112 ZB132 ZB152 ZB262 ZB272 ZB332 ZB392 ZC032 ZC352 ZC552 4C085 AA13 AA14 BB11 EE01 4C087 AA01 AA02 BC83 MA02 NA13 NA14 ZA01 ZA02 ZA15 ZA16 ZA36 ZA38 ZA51 ZA53 FA75 ZA74 FA75 FA75 FA75 ZA72 FA75 ZA74 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A5 A3

Claims (23)

[Claims] 1. An isolated nucleic acid molecule, which comprises: (a) a polynucleotide fragment of SEQ ID NO: X, or a polynucleotide fragment of the cDNA sequence contained in ATCC Deposit No. Z that is hybridizable to SEQ ID NO: X. (B) a polynucleotide encoding a polypeptide fragment of SEQ ID NO: or a polypeptide fragment encoded by the cDNA sequence contained in ATCC Deposit No. Z capable of hybridizing to SEQ ID NO: X; (c) SEQ ID NO: Y; Or a polynucleotide encoding a polypeptide domain encoded by a cDNA sequence contained in ATCC Deposit No. Z capable of hybridizing to SEQ ID NO: X; (d) a polypeptide epitope of SEQ ID NO :, or a sequence Hybrida on number X Polynucleotide encoding the polypeptide epitope encoded by the cDNA sequence contained in ATCC Deposit No. Z; (e) a polynucleotide encoding a polypeptide of SEQ ID NO: Y having biological activity, or a sequence CDNA sequence contained in ATCC Deposit No. Z capable of hybridizing to number X; (f) Polynucleotide that is a variant of SEQ ID NO: X; (g) Polynucleotide that is an allelic variant of SEQ ID NO: X; (h) A polynucleotide encoding a species homologue of SEQ ID NO: (i) a polynucleotide capable of hybridizing to any one of the polynucleotides specified in (a) to (h) under stringent conditions, Here, the polynucleotide has a nucleotide sequence consisting of only A residues or 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 the polynucleotide fragment comprises a nucleotide sequence encoding a secreted protein. 3. The polynucleotide fragment comprises a sequence identified as SEQ ID NO: Y, or a nucleotide sequence encoding a polypeptide encoded by the cDNA sequence contained in ATCC Deposit No. Z, which is capable of hybridizing to SEQ ID NO: X. 2. The isolated nucleic acid molecule of claim 1, which comprises. 4. The isolated of claim 1, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO: X, or the cDNA sequence contained in ATCC Deposit No. Z capable of hybridizing to SEQ ID NO: X. Nucleic acid molecule. 5. The isolated nucleic acid molecule of claim 2, wherein the nucleotide sequence comprises consecutive nucleotide deletions from either the C-terminus or the N-terminus. 6. The isolated nucleic acid molecule of claim 3, wherein the nucleotide sequence comprises consecutive nucleotide deletions 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 of making 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, which comprises 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; (b) a biological activity. (C) a polypeptide domain 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 Deposit No. Z; d) a polypeptide epitope of the encoded sequence contained in SEQ ID NO: Y, or ATCC deposit number Z; (e) a secreted form of the encoded sequence contained in SEQ ID NO: Y, or ATCC deposit number Z; ) SEQ ID NO: Y, or the full length protein of the encoded sequence contained in ATCC Deposit No. Z; (g) a variant of SEQ ID NO: Y (h) 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,
Isolated polypeptide. 12. The isolated polypeptide of claim 11, wherein the secreted form or the full-length protein comprises a contiguous amino acid deletion from either the C-terminus or the 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 cell. 15. A method of producing an isolated polypeptide, comprising the step of: (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 of preventing, treating or alleviating a medical condition, comprising:
13. A method comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim 11 or the polynucleotide of claim 1. 18. A method for diagnosing a pathological condition or susceptibility to a pathological condition in a subject, comprising the step of: (a) determining the presence or absence of a mutation in the polynucleotide of claim 1. And (b) diagnosing a pathological condition, or susceptibility to a 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 or amount of expression of the polypeptide of claim 11 in a biological sample. And (b) diagnosing a pathological condition, or susceptibility to a pathological condition, based on the presence or amount of expression of the polypeptide. 20. A method of identifying a binding partner for the polypeptide of claim 11, comprising: (a) contacting the polypeptide of claim 11 with a binding partner;
And (b) determining whether the binding partner results in activity of the polypeptide. 21. A gene corresponding to the cDNA sequence of SEQ ID NO: Y. 22. A method of identifying activity in a biological assay, comprising:
Wherein the method comprises: (a) expressing SEQ ID NO: X in cells; (b) isolating the supernatant; (c) detecting activity in a biological assay; and (d) ) Identifying a protein in the supernatant that has the activity. 23. A product produced by the method of claim 20.