New cytokine zalpha 11-ligand

FIELD: biotechnology, medicine.

SUBSTANCE: Zalpha 11-ligand is isolated from cDNA library generated from activated cells of human peripheral blood that have been selected from CD3. Animal is inoculated with Zalpha 11-ligand and antibodies are prepared that are able to bind specifically with epitopes, peptides or polypeptides of Zalpha 11-ligand. Invention provides effective regulation and/or development of hemopoietic cells in vitro and in vivo. Invention can be used for preparing Zalpha 11-ligand and antibodies for it.

EFFECT: valuable properties of new cytokine.

18 cl, 5 tbl, 1 dwg, 55 ex

 

BACKGROUND of the INVENTION

Proliferation and differentiation of cells of multicellular organisms are regulated by hormones and polypeptide growth factors. These diffundere molecules allow cells to communicate with each other and act together to form cells, tissues and organs and repair damaged tissue. Examples of hormones and growth factors include steroid hormones (e.g. estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, interleukins, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.

Hormones and growth factors affect cell metabolism by binding with receptors. Receptors can be integral membrane proteins that are associated with the transmission of signals in the cell, such as a system of second messengers. Other classes of receptors are soluble molecules such as transcription factors.

Cytokines usually stimulate the proliferation and/or differentiation of cells of hematopoietic lines of differentiation or participate in the mechanisms of immune and inflammatory reactions. Examples of cytokines that affect hematopoiesis, are erythropoietin (EPO), which stimulates R is sweetie erythrocytes; thrombopoetin (TRO), which stimulates cell growth megakaryocytes line of differentiation; and granulocyte colony-stimulating factor (G-CSF), which stimulates the growth of neutrophils. These cytokines are applicable in the restoration of normal levels of blood cells in patients suffering from anemia, thrombocytopenia and neutropenia or receiving chemotherapy for cancer.

Interleukins are a family of cytokines, which mediate the immune response, including inflammation. Interleukins mediate a variety of inflammatory pathology. Central to the immune response is a T-cell, which produces many cytokines and artificial (acquired) immunity to antigens. Cytokines produced by T-cells were classified as cytokines of type 1 and type 2 (Kelso, A. Immun. Cell Biol. 76:300-317, 1998). The type 1 cytokines include IL-2, IFN-γ, LT-αand participate in inflammatory reactions, antiviral immunity, immunity against intracellular parasites and graft rejection. The type 2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13 and are involved in humoral responses, anthelminthic immunity and allergic reactions. Common cytokines between type 1 and type 2 include IL-3, GM-CSF and TNF-α. There is evidence in favor of the assumption that the population of T cells, the product is yousie cytokines of type 1 and type 2 mainly migrate to different types of inflamed tissue.

Mature T cells can be activated, namely the antigen or other incentive to produce, for example, cytokines, molecules of biochemical signaling or receptor, which also influence the fate of populations of T cells.

B cells can be activated through receptors on their cell surface, including the receptor for b cells, and other accessory molecules to perform auxiliary cellular functions, such as production of cytokines.

Natural killer cells (m-cells) have a common cell precursor T-cells and b-cells and play a role in the immunological control. NK-cells, which contain up to 15% of blood lymphocytes that do not Express receptors for antigens and, therefore, do not use MNF-recognition as a requirement for binding to cell-target. NK cells are involved in the recognition and killing certain tumor cells and virus-infected cells. It is considered that, in vivo MK-cells require activation, but it has been shown that in vitro MK-cells kill some types of tumor cells without activation.

Demonstrated in vivo activity of a family of cytokines illustrate the enormous clinical potential of, and need for other cytokines, agonists of cytokines and antagonis the s cytokines. This invention is directed to meeting these needs by providing a new cytokine that stimulates cells of hematopoietic lines of differentiation, as well as related compositions and methods.

This invention provides such polypeptides for these and other applications, which should be obvious to persons of ordinary skill in this field from here.

The drawing is an illustration of multiple mappings IL-2, IL-15 human zalpha11 ligand (SEQ ID NO:2), IL-4 human IL-4-mouse GM-CSF human GM-CSF mouse.

DETAILED description of the INVENTION

Before a detailed description of the present invention may be useful for understanding the definition of the following terms:

The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to the second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to the substrate. In principle, any peptide or protein to the antibody or other specific binding agent may be used as affinity tags. Affinity tags include polyhistidine area (tract), protein a (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzvmol. 198:3, 1991), glutathione S-t is ansfers (Smith and Johnson, Gene 67:31, 1988), affinity tag, Glu-Glu (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P, Flag peptide™ (Hopp et al., Biotechnology 6:1204-10, 1988), strategicinitiatives peptide, or other antigenic epitope or binding domain. See, in General, Ford et al., Protein Expression and Purification 2:95-107, 1991. DNA encoding affinity tags that are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ).

The term "allelic variant" is used here to denote any of the two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation occurs through natural mutation and can lead to phenotypic polymorphism in populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is used here to refer to the proteins encoded by allelic variant of a gene.

The terms "amino-terminal" and "carboxyl-terminal" are used here to denote positions within the polypeptide. Where allows context, these terms are used with reference to a specific sequence or part of a polypeptide to indicate proximity or relative position. For example, a certain sequence, located carboxyl-terminal relative to siloc the second sequence in the polypeptide, located proximally relative to carboxyl-end reference sequence, but not necessarily located at carboxyl-end of the full polypeptide.

The term "pair complement/anticomplement" denotes non-identical parts of molecules, which form ecovalence associated, stable couple in suitable conditions. For example, Biotin and avidin (or streptavidin) are members-prototypes pairs complement/anticomplement. Other examples of pairs complement/anticomplement include a pair of receptor/ligand pairs antibody/antigen (or hapten, or spitup), a pair of sense/antisense polynucleotide, etc. In the case where it is desired subsequent dissociation of pairs complement/anticomplement, a pair of complement/anticomplement preferably has a binding affinity of <109M-1.

The term "compliments polynucleotide molecule" refers to a polynucleotide molecule having a complementary sequence of bases and inverted orientation compared with the reference sequence. For example, the sequence 5'-ATGCACGGG-3' complementary to the 5'-CCCGTGCAT-3'.

The term "degenerate nucleotide sequence" means a sequence of nucleotides that includes one or more degenerate codons (as compared with a reference polynucleotide molecule that to the range polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e. triplets GAU and GAC, each encode Asp).

The term "expressing vector" is used to refer to a DNA molecule, linear or circular, which contains a segment that encodes an polypeptide that is functionally linked to additional segments that provide for its transcription. Such additional elements include a promoter and termination sequences, and may also include one or more start points of replication, one or more breeding markers, enhancer, polyadenylation signal, etc. Expressing vectors usually made from plasmid or viral DNA, or may contain elements of both.

The term "isolated" as applied to polynucleotide indicates that this polynucleotide has been removed from its natural genetic environment and, therefore, does not contain other extraneous or unwanted coding sequences, and is in a form suitable for use in genetically-engineered systems for the production of proteins. These selected molecules are molecules that are isolated from their natural environment and include cDNA clones and genomic clones. The selected DNA molecules given the CSOs of the invention do not contain other genes, they are usually associated, but may include naturally occurring 5'- and 3'-noncoding regions, such as promoters and terminators. Identification of related areas will be obvious to a person of ordinary skill in the art (see, for example, Dynan and Tijan, Nature 316:774-78, 1985).

"Isolated" polypeptide or protein is a polypeptide or protein, which is found in conditions other than its natural environment, such as apart from blood and animal tissue. In a preferred form, the selected polypeptide is essentially not containing other polypeptides, particularly other proteins of animal origin. Preferably provision of polypeptides in high purity form, i.e. having a purity of more than 95%, more preferably a purity of more than 99%. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternative glycosylated or derivationally form.

The term "neoplastic" when applied to cells indicates cells exposed to new and aberrant proliferation, particularly in tissues where this proliferation is uncontrolled and progressive, leading to neoplasms (tumors, i.e. tumors). Neoplastic (related to the tumor the cells can be either malignant, i.e. invasive and metastatic or benign.

The term "functionally (resectable) related", when referring to DNA segments, indicates that these segments are arranged so that they operate together for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the coding segment to the terminator.

The term "ortholog" refers to a polypeptide or protein obtained from one species that is the functional copy of the polypeptide or protein from a different species. Differences in sequence among the orthologues are the result of speciation.

"Paralogy" are different, but structurally related proteins produced by the body. It is believed that paralogy arise from gene duplication. For example, α-globin, β-globin and myoglobin are paralogue each other.

The term "polynucleotide" means a single - or double-stranded polymer of deoxyribonucleotide or ribonucleotidic bases read from the 5'-end to 3'-end. Polynucleotide include RNA and DNA can be isolated from natural sources, synthesized in vitro or obtained from a combination of natural and synthetic molecules. The size of polynucleotides are expressed as pairs of nucleotides (abbreviated as "BP"), nucleotide ("NT") or thousands of pairs of nucleotide the ("TPN"). Where allows context, the last two terms can describe polynucleotide, which are single-stranded or double-stranded. When applying this term to the double-stranded molecules it is used to denote the total length, and it should be clear that it is equivalent to the term "nucleotide pairs". Specialists in this field will be clear that the two strands of double-stranded polynucleotide may differ slightly in length and that their ends can be arranged in zigzags in the enzymatic degradation; thus, not all of the nucleotides in the double-stranded polynucleotide molecule can be paired.

"Polypeptide" is a polymer of amino acid residues connected by peptide bonds, if he by natural or synthetic means. Polypeptides having less than about 10 amino acid residues, usually referred to as "peptides".

The term "promoter" is used here in its recognized in this field the value to denote a part of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are detected usually, but not always, in the 5'non-coding regions of genes.

"Protein" refers to a macromolecule containing one or more polypeptide chains. Protein can in order to include ones components, such as carbohydrate groups. Carbohydrates and other ones of the components may be attached to a protein by the cell producing the protein, and will vary depending on the cell type. Proteins are defined here in terms of their amino acid structures; alternates, such as carbohydrate groups, usually not specified, but nevertheless, they may be present.

The term "receptor" is used here to denote the associated cell protein that binds to a bioactive molecule ("ligand") and Mediaset the effect of this ligand on the cell. Membrane-bound receptors are characterized by multipeptide structure containing the extracellular ligand binding domain and an intracellular domain that is typically involved in signal transmission. The binding of ligand to the receptor causes a conformational change in the receptor that causes the interaction between the effector domain (domains) and another molecule (molecules) in the cell. These interactions, in turn, lead to changes in cell metabolism. Metabolic events associated with the interactions of receptor-ligand include gene transcription, phosphorylation, dephosphorylation, increasing the production of cyclic AMP, mobilization of cellular calcium, mobilization of membrane lipids, cellular edges the Yu, hydrolysis of inositides and hydrolysis of phospholipids. Typically, the receptors can be membrane-bound, cytosolic or nuclear; Monomeric (e.g., the receptor for thyroid-stimulating hormone receptor β-adrenergic hormone) or multimeric (e.g., PDGF-receptor, the receptor for growth hormone, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6-receptor).

The term "secretory signal sequence" refers to the sequence of DNA which encodes a polypeptide ("secretory polypeptide"), which as a component of a larger polypeptide, directs this larger polypeptide through a secretory pathway cells in which it is synthesized. This larger polypeptide usually split with the destruction of secretory peptide during passage through the secretory pathway.

The term "splicing variant" is used here to refer to alternative forms of RNA transcribed from the gene. Splicing variation occurs through natural use of alternative splicing sites in the transcribed RNA molecule or less usually between separately transcribed RNA molecules and can lead to multiple mRNA transcribed from the same gene. Splicing variants may encode polypeptides having altered amino acid serial is lnost. The term splicing variant used here to refer to the protein encoded splicing variant mRNA transcribed from the gene.

It should be clear that the molecular weight and length of the polymers, determined inaccurate analytical methods (e.g., gel electrophoresis), are approximate values. When this value is expressed as "about" or "approximately" X, the value X should be understood as defined in accuracy ±10%.

All references cited here included by reference in their entirety.

This invention is based in part on the discovery of a new sequence of DNA which encodes a protein with a structure having chetyrehspalnyh bundle cytokine. Through the process of cloning, analysis of cell proliferation and research associate, described in detail here, was identified polynucleotide sequence encoding a new ligand polypeptide, i.e. a ligand with high specificity with respect to the previously described orphan ("orphan")-zalpha11 receptor. This polypeptide ligand, named zalpha11-ligand, was isolated from a cDNA library generated from activated cells of peripheral blood (hPBCs), which were selected on CD3. CD3 is a marker of cell surface that are unique to cell lymphoid the CSOs origin, in particular, T cells.

In the following examples, the cell line that is dependent on related orphan receptor zalpha11 the way for the survival and growth of cells in the absence of other growth factors used for screening the cDNA source coding zalpha11 ligand. Preferred dependent on growth factors cell line used for transfection and expression of zalpha11 receptor was line BaF3 (Palados and Steinmetz, Cell 41:727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6:4133-4135, 1986). However, other dependent growth factors cell lines, such as FDC-P1 (Hapel et al., Blood 64:786-790, 1984) and M07e (Kiss et al., Leukemia 7:235-240. 1993), are suitable for this purpose.

Amino acid sequence for the zalpha11 receptor showed that it encoded a receptor belongs to the subfamily of receptors of cytokines of Class I, which includes, but is not ogranichivaya them, the receptors for IL-2, IL-4, IL-7, IL-15, EPO, TPO, GM-CSF and G-CSF (for a Review, see Cosman, "The Hematopoietin Receptor Superfamily" in Cytokine 5(2):95-106, 1993). Zalpha11 receptor fully described in the patent application PCT joint ownership with the number US99/22149. Analysis of tissue distribution of mRNA zalpha11 receptor showed expression in lymph node, peripheral blood leukocytes (PBL), spleen, bone marrow and thymus (thymus). In addition, this mRNA was abundant in the cell line Raji (ATCC No. CCL-86), derived from a Burkitt lymphoma. TKA is Evoe distribution of this receptor suggests, what targets predicted zalpha11 ligand are cells of hematopoietic origin, particularly lymphoid precursor cells and lymphoid cells. Other having a structure chetyrekhmernogo beam cytokines that act on lymphoid cells include IL-2, IL-4, IL-7 and IL-15. In respect of the review has a structure chetyrekhmernogo beam of cytokines, see Nicola et al., Advances in Protein Chemistry 52:1-65, 1999, and Kelso, A., Immunol. Cell BjoL76:300-317, 1998.

The air-conditioned environment (COP) are selected CD3+, PMA/Ionomycin-stimulated peripheral blood cells of a person supported the growth of BaF3 cells that expressed zalpha11 receptor and were otherwise dependent on IL-3. Conditioned medium from cells that were not 1) PMA/Ionomycin-stimulated; and 2) CD3 selected (with PMA/Ionomycin stimulation or without it), did not support the growth of cells BaF3/zalpha11 receptor. Control experiments showed that the proliferative activity was not associated with other known growth factors, and that the ability of such conditioned media to stimulate the proliferation of expressing zalpha11 receptor cells could be neutralized soluble form of this receptor.

The proliferation of zalpha11 receptor-expressing BaF3 cells exposed to CS (conditioned medium) from CD3+ selected FMA/Eonomic the n-stimulated peripheral blood cells of a person, identified by visual inspection of crops and/or analysis of proliferation. Numerous suitable proliferation assays known in this area and they include tests for reduction of the dye, such as alamarBlue™ (AccuMed International, Inc. Westlake, Ohio), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (Mosman, J. Immunol. Meth. 65:55 to 63. 1983); 3- (4,5-dimethylthiazol-2-yl)-5-3-carboxymethoxy-2H-tetrazole; 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-teratological and caneditallcategories (which are commercially available from Polysciences, Inc., Warrington, PA); analyses of mitogenesis, such as, for example, measurement include3H-thymidine; analyses of displacement of the dye, using, for example, dyes, naphthalene black or tripney blue; the absorption of the dye using decamillionaire; and the release of chromium. Cm. in General, Freshney, Culture of Animal Cells: A Manual of Basic Technique. 3rded., Wiley-Liss, 1994, incl. here as a reference.

The cDNA library was obtained from CD3+ selected PMA/Ionomycin-stimulated peripheral blood cells of man. The cDNA library CD3+ selected PMA/Ionomycin-stimulated cells in human peripheral blood was divided into pools containing multiple cDNA molecules, and transfusional in line host cells, for example, in cells KSS 570 (ATSS accession no. 10314). Cord is lirovannye cells-the owners were cultured in the medium, which did not contain exogenous growth factors, and conditioned medium was collected. Conditioned medium was analyzed for its ability to stimulate the proliferation of BaF3 cells, transfected with the zalpha11 receptor. Were identified pools of cDNA, producing air-conditioned environment, which stimulated cells BaF3/zalpha11 receptor. This combined plasmid cDNA was electroporative in E. coli. cDNA was isolated from single colonies and transfusional individually in cells KSS 570. Positive clones were identified by positive results in the analysis of cell proliferation Var/zlh11-receptor and specificity were tested by neutralization of proliferation to soluble zalpha11 receptor.

Positive clones were isolated, and the sequencing analysis revealed that the polynucleotide sequence contained in this plasmid DNA was new. The secretory signal sequence comprises amino acid residues 1 (Met) - 31 (Gly), and the Mature polypeptide consists of amino acid residues 32 (Gln) to 162 (Ser) as shown in SEQ ID NO:2).

Usually it is predicted that cytokines have a structure consisting of four alpha-helices and helix a, C, and D are the most important in the interactions of the ligand-receptor and are more highly conserved among members of this family. With reference to aminokislotnoi sequence zalpha11 ligand person, shown in SEQ ID NO:2, a comparison of the amino acid sequence zalpha11 ligand human IL-15 human IL-4 and GM-CSF person, it is projected that the spiral And zalpha11 ligand is defined by amino acid residues 41-56; spiral amino acid residues 69-84; spiral With amino acid residues 92-105 and helix D amino acid residues 135-148; shown in SEQ ID NO:2. Structural analysis suggests that the a/b loop is long, In/S-loop is short and the C/D loop is parallel to the long. This structure of loops leads to a spiral organization up-up-down-down. Residues of cysteine are absolutely conservative between zalpha11-ligand and IL-15, as shown in the drawing. Residues of cysteine, which are conservative between IL-15 and zalpha11-ligand correspond to amino acid residues 71, 78, 122 and 125 of SEQ ID NO:2. Save some cysteine residues were also found in IL-2, IL-4, GM-CSF and zalpha11-ligand corresponding to amino acid residues 78-125 SEQ ID NO:2, as shown in the drawing. Matching location cysteine is an additional confirmation of the structure chetyrekhmernogo beam. Also highly conserved in this family, containing IL-15, IL-2, IL-4, GM-CSF and zalpha11 ligand is the sequence Glu-Phe-Leu shown in SEQ ID NO:2 from residues 136-138 in the drawing.

Subsequent analysis of zalpha11 ligand on cos the ve multiple comparisons (shown in the drawing) predicts, what amino acid residues 44, 47 and 135 (shown in SEQ ID NO:2) play an important role in linking zalpha11 ligand with its cognate receptor. In addition, the predicted amino acid sequence of murine zalpha11 ligand finds 57% identity with the predicted human protein. On the basis of sequence comparison zalpha11 ligand of human and mouse well preserved remains were found in areas that are predicted to encode alpha-helices a and D. the Corresponding polynucleotide coding regions, domains, motifs, residues and sequences zalpha11 ligand described herein, is shown in SEQ ID NO:1.

Detailed mutational analysis was performed for IL-4 and IL-2, both of which are vysokoreaktsionnye zalpha11-ligand. Analysis of murine IL-2 (Zurawski et al., EMBO J. 12:5113-5119, 1993) shows that the residues in helices a and C are important for binding to IL-2Rβ; critical residues are Asp34, Asn99and Asn103. Numerous remains in the loop And/murine IL-2 and spiral are important for binding to IL-2Rαwhile only a single residue, Gln141in helix D is very important for binding to IL-2Rα. Similarly, helix a and C are sites of interaction between IL-4 and IL-4Rα (structurally similar to IL-2Rα), and residues in helix D are essential for the interaction of IL-2Rα (Wang t al., Proc. Natl. Acad. Sci. USA 94:1657-1662, 1997; Kruse et al., EMBQ.J. 11:3237-3244, 1992). In particular, mutation of Tyr124in Asp IL-4 person creates antagonist that binds to IL-4Rαbut not with IL-2Rα and, therefore, cannot transmit a signal (Kruse et al. ibid. 1992).

While the spiral And is relatively well preserved between human and mouse zalpha11-ligand, the coil is more divergent. Although both species are predominant acidic amino acids in this region, these differences may be responsible for the species specificity of the interaction between zalpha11-ligand and its receptor "β"-type of zalpha11. Loop a/b and spiral In zalpha11 ligand are quite conservative among species; although subunit of the receptor corresponding to the IL-2Rα, has not yet been identified, the conservatism in this area suggests that it is functionally important. D-helix of human and mouse zalpha11 ligand are also highly conserved. Antagonists zalpha11 receptor can be constructed through mutations in helix D zalpha11 ligand. They may include shortening of this protein from residue Gln145(SEQ ID NO:2) or mutation of Gln145or lle148(SEQ ID NO:2; the corresponding Tyr124in IL-4 man) residues, such as Ala or Asp. Any mutation that disrupts the helical structure zalpha11 ligand, can eliminate svyazyvaniye receptor and, therefore, to inhibit the transmission of the signal.

Cytokines structure chetyrekhmernogo beam group also along the length of their components-helices. Cytokines "linopirdine forms usually consist of spirals from 24-30 residues and include IL-6, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF) and human growth hormone (hGH). Cytokines "korotkoperiodnoi forms usually consist of helices from residues 18-21 and include IL-2, IL-4 and GM-CSF. The authors believe that zalpha11 ligand is a new member of the group of cytokines korotkoperiodnoi form. Studies using CNTF and IL-6 showed that the spiral CNTF can replace the equivalent helix in IL-6, giving the Chimera properties of binding of CNTF. Thus, functional domains chetyrehspalnyh cytokines, apparently, are determined on the basis of structural homology, regardless of the identity of the sequence and can maintain the functional integrity in the Chimera (Kallen et al., J. Biol. Chem. 274:11859-11867, 1999). Thus, the helical domains zalpha11 ligand will be applicable for obtaining chimeric fused molecules, in particular, with other cytokines korotkoperiodnoi form, to determine and modulate the binding specificity of the receptor. Of particular interest are fused (hybrid) protein, designed with spiral and/or helix D, or slit the tree, which unite spiral and loop domains of other cytokines korotkoperiodnoi forms, such as IL-2, IL-4, IL-15 and GM-CSF. Amino acid residues comprising helix a, b, C and D and loop A/b, b/S and C/D for zalpha11 ligand, IL-2, IL-4, IL-15 and GM-CSF are shown in table 1.

Table 1
SpiralLoopSpiralLoopSpiralLoopSpiralD
AndA/bInIn/SC/D
The remains of zalpha11 ligand41-5657-6869-8485-9192-105106-134135-148SEQ ID no:2
The remains of IL-236-4647-5253-7576-8687-99100-102103-121SEQ ID no:111
The remains of IL-429-4344-6465-8384-9495-118119-133134-151SEQ ID no 112
The remains of IL-1545-6869-8384-101102 to 106107-119 120-133134-160SEQ ID no 113
The remains of GM-CSF30-4445-7172-8182-9091-102103-119120-131SEQ ID no 114

This invention provides polynucleotide molecules, including DNA and RNA that encode the polypeptides described herein zalpha11 ligand. Specialists in this field will be clear that due to the degeneracy of the genetic code, there can be significant variation among these polynucleotide molecules. SEQ ID NO:3 is a degenerate DNA sequence that encompasses all DNAs that encode the polypeptide zalpha11 ligand SEQ ID NO:2. Specialists in this field will be clear that the degenerate sequence of SEQ ID NO:3 also provides all RNA sequences encoding SEQ ID NO:2, by substituting U (uracil) T (thymine). Thus, encoding the polypeptide zalpha11 ligand polynucleotide containing the region from nucleotide 1 or 97 to nucleotide 486 SEQ ID NO:3, and their RNA equivalents are considered by the present invention. Table 2 gives the one-letter codes used within SEQ ID NO:3 to refer to the provisions of degenerate nucleotides. Permissions represent the nucleotides denoted by a code letter. "Complement" indicates the code for the complementary nucleotide sequence that is Chida (complementary nucleotides). For example, the code Y denotes either C or T, and its complement R denotes a or G, and a is complementary to T and G is complementary to C.

TABLE 2
NucleotideResolutionComplementResolution
AndAndTT
GG
GG
TTAndAnd
RA|GYC|T
YC|TRA|G
MA|CToG|T
ToG|TMA|C
SC|GSC|G
WA|TWA|T
NA|s|TDA|G|T
InC|G|TVA|C|G
VA|C|GInC|G|T
DA|G|TNA|C|T
A|C|G|TNA|C|G|T

Degenerate codons used in SEQ ID NO:3, which includes all possible codons for a particular amino acid are presented in table 3.

TABLE 3
Amino acidSingle-letter codeCodons codonDegenerate
CysTGC TGTTGY
SerSAGC AGT TCA TCC TCG TSCWSN
ThrTACA ACC ACG ACTACN
ProPCCA CCC CCG CCTCCN
AlaAndGCA GCC GCG GCTGCN
GlyGGGA GGC GGG GGTGGN
AsnNAAS AATAAY
AspDGAC GATGAY
GluEGAA GAGGAR
GlnQCAA CAGCAR
HlsNCAC CATCAY
ArgRAGA AGG CGA CGC CGG CGTMGN
LysToAAA AAGAAR
MetMATGATG
IleIATA ATC ATTATH
LeuLOne HUNDRED CTC CTG CTT TTA TTGYTN
ValVGTA GTC GTG GTTGTN
PheFTTC TTTTTY
TightYTAC TATTAY
TrpWTGGTGG
Ter.TAA TAG TGATRR
Asn|AspInRAY
Glu|GlnZSAR
AnyXNNN

The person of ordinary skill in this field will be clear that some ambiguity is introduced in the definition of degenerate codon representing all possible codons encoding each amino acid. For example, the degeneracy of the codon for serine (WSN) may in some circumstances to encode arginine (AGR), and the degeneracy of the codon for arginine (MGN) may in some circumstances to encode serine (AGY). A similar relationship to what exists between codons, encoding phenylalanine and leucine. Thus, some polynucleotides covered by the degenerate sequence may encode variant amino acid sequences, but the person of ordinary skill in this field can easily identify such variant sequences by comparison with the amino acid sequence of SEQ ID NO:2. Variant sequences can be readily tested for functionality as described herein. Thus, the proposed polynucleotide molecule where these nucleotides are as shown in SEQ ID NO:3 from nucleotide 121 to nucleotide 444 or from nucleotide 94 to nucleotide 486.

The person of ordinary skill in this field will also be clear that the different types can be preferred (preferencyjne) using codons". In General, see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al., Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13: 355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm. Nuc. Adds Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. In the application herein, the term "preferred (preferencyjne) using codons" or "preferred (preferencyjne) codons" is the term for this area related to the codons of protein translation that is most frequently used in the cells of certain species, giving, therefore, a preference for one or few p is estuvieran possible codons, encoding each amino acid (See. table 3). For example, the amino acid threonine (Thr) can be encoded ACA, ACC, ACG, or ACT, but in mammals the most commonly used codon is ACC; in other species, for example, insect cells, yeast, viruses or bacteria, may be preferred other Thr codons. Preferred codons for a particular species can be introduced into polynucleotide of the present invention in a variety of ways known in this field. Introduction the preferred sequence of codons in recombinant DNA may, for example, to increase the production of this protein, making more efficient protein translation in a particular cell type or form. Thus, the degenerate sequence of codons described in SEQ ID NO:3, serves as a matrix for optimization of expression of polynucleotides in various cell types and species commonly used in this field and are described here. Sequences that contain preferred codons can be tested and optimized for expression in different species and tested for functionality as described herein.

As previously noted, the selected polynucleotide of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known in this field. Typically, RNA is extracted from tissue or cells, the which produces large quantities of RNA zalpha11 ligand. Such tissues and cells identify Northern-blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980) or by screening, air-conditioned environment from different types of cells on the activity of the cells or target tissues. As soon as this activity or producing RNA cell or tissue is identified, the total RNA can be obtained using extraction isothiocyanato guanidine, followed by separation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly(A)+RNA is obtained from the total RNA using the method of Aviv and Leder, Proc. Natl. Acad. Sci. USA 69:1408-1412 (1972). Complementary DNA (cDNA) obtained from the floor(A)+RNA using known methods. Alternatively, it may be isolated genomic DNA. Then polynucleotide encoding polypeptides zalpha11 ligand, identify and distinguish, for example, using hybridization or polymerase chain reaction (PCR).

Full size clone encoding zalpha11 ligand, can be obtained by conventional cloning procedures. Clones complementary DNA (cDNA) are preferred, although for some applications (for example, expressive in transgenic animals) may be preferable to use a genomic clone or modification of the cDNA clone to include at least one genomic intron. Methods for obtaining clones of cDNA and genomic clone of the well known and are within the ordinary skill in this field include the use described here, the sequence or its parts for probing or priming of the library. Expression libraries can be probed with antibodies to fragments zalpha 11-receptor or other specific binding partners.

Polynucleotide sequence zalpha11 ligand described herein can also be used as probes or primers to clone 5'-non-coding regions of the gene zalpha11 ligand. In connection with tissue-specific expression observed for zalpha11 ligand, it is expected that this area of the gene provides the hematopoiesis-specific and lymphoid-specific expression. Thus, the promoter elements of the gene zalpha11 ligand could direct tissue-specific expression of heterologous genes in, for example, transgenic animals or patients subjected to gene therapy. Cloning of 5'flanking sequences also facilitates obtaining proteins zalpha11 ligand "gene activation"as described in U.S. patent US Patent No. 5641670. Briefly, expression of an endogenous gene zalpha11 ligand in the cell change introduction in the locus zalpha11 ligand design DNA containing at least a targeting sequence, a regulatory sequence, an exon, and an unpaired donor site of splicing. The target sequence is 5'-non-coding sequence zalpha11 ligand, which makes possible homologous recombination of this design with endogen the m locus zalpha11 ligand, whereby the sequences within the construct become functionally linked to the endogenous coding zalpha11 ligand sequence. Thus endogenous promoter zalpha11 ligand can be replaced or supplemented with other regulatory sequences to provide enhanced tissue-specific, or otherwise regulated expression.

Further, this invention provides copies of polypeptides and polynucleotides from other species (orthologues). These species include, but are not limited to, mammals, birds, amphibians, reptiles, fishes, insects, and other species of vertebrates and invertebrates. Of particular interest are polypeptides zalpha11 ligand from other mammalian species, including polypeptides mice, pigs, sheep, cow, dog, cat, horse species and polypeptides other primates. Orthologues zalpha11 ligand person can be cloned using information and compositions provided by the present invention in combination with conventional methods of cloning. For example, cDNA can be cloned using mRNA obtained from a tissue type or cell that expresses zalpha11 ligand, as described herein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from opican the x sequences here. Then prepare a library from mRNA positive tissue or cell line. Then the coding zalpha11 ligand cDNA can be isolated in various ways, such as sensing full or partial human cDNA or with one or more sets of degenerate probes based on the sequences described. This cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4683202), using primers designed as described here is typical sequence zalpha11 ligand person. In an additional method, the cDNA library can be used for transformation or transfection of host cells, and expression of the cDNA of interest can be detected with an antibody to the polypeptide zalpha11 ligand, research associate or analysis activity. Such methods can be applied also to highlight genomic clones.

The polynucleotide sequence for the mouse ortholog of zalpha11 ligand was identified and is shown in SEQ ID NO:55, and the corresponding amino acid sequence shown in SEQ ID NO:56. There is a 62% identity between mouse and human sequences for the district of 124 amino acids, which corresponds to residues 30-153 in SEQ ID NO:2 and residues 23-146 SEQ ID NO:56 zalpha11 ligand. Mature posledovatelno the ü for murine zalpha11 ligand presumably begins when His 18(as shown in SEQ ID NO:56), which corresponds to His25(as shown in SEQ ID NO:2) in the human sequence. As a shortened form of the human polypeptide is active, it seems likely that the equivalent of the polypeptide of mouse zalpha11 ligand (i.e. without rests His18-Pro22SEQ ID NO:56) is also active. Tissue analysis revealed that the expression of murine zalphall-ligand found in testis, spleen and thymus (thymus).

Specialists in this field will be clear that the sequence described in SEQ ID NO:1 represents a single allele of a zalpha11 ligand person and it is expected that the existence of allelic variation and alternative splicing. Allelic variants of this sequence can be cloned by probing cDNA libraries or genomic libraries from different individuals in accordance with standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1, including variants containing silent mutations and variants in which mutations lead to changes in amino acid sequence, are in the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO:2. cDNA generated from educated alternative splicing of mRNAs, which retain the properties of the polypeptide zalpha11, included in the scope of the present invention, as are polypeptides encoded by these cDNA and mRNA. Allelic variants and splicing variants of these sequences can be cloned by probing cDNA libraries or genomic libraries from different individuals or tissues according to standard procedures known in the field. Thus, the proposed polynucleotide molecule where these nucleotides are as shown in SEQ ID NO:1 from nucleotides 167 to nucleotide 490 or from nucleotide 140 to nucleotide 532.

Gene zalpha11 ligand was mapped relative to the marker SHGC-12342 frame IL-2 location zalpha11 ligand at a distance of 180 TPN from this marker IL-2. The use of surrounding markers puts gene zalpha11 ligand in the 4q27 region on the map of the integrated LDB chromosome 4 (The Genetic Location Database, University of Southhampton). The invention also provides reagents which will find use in diagnostic applications. For example, gene zalpha11 ligand probe containing DNA or RNA zalpha11 ligand or suppositionally, can be used to determine whether gene zalpha11 ligand on the chromosome, such as chromosome 4, or whether there was a gene mutation. Based on the annotation of a fragment of human genomic DNA containing part of the genomic DNA zalpha11 ligand (Genbank Accession No. (gene Bank access) is S), zalpha11 ligand localized in the 4q27 region of chromosome 4. Detected chromosomal aberrations in the gene locus zalpha11 ligand include, but are not limited to, aneuploidy, changes in the copy number of the gene, loss of heterogeneity (LOH), translocations, insertions, deletions, and changes to the sites of the restriction and rearrangement. Such aberrations can be detected using polynucleotides of the present invention by application of molecular genetic methods, such as analysis of length polymorphism restriction fragments (RFLP)analysis, short tandem repeats (STR) using PCR methods, and other methods of genetic analysis of coupling known in the art (Sambrook et al., ibid; Ausubel et al., ibid; Marian, Chest 108:255-65, 1995).

Exact knowledge of the position of a gene may be useful for a number of purposes, including: 1) determine whether the sequence is part of an existing reference, and more surrounding genetic sequences in various forms, such as YAC, YOU or cDNA clones; 2) provide a possible gene candidates for hereditary diseases, which detects the clutch with the same chromosomal region; and 3) cross-comparison with model organisms such as the mouse, which may help to identify which function may have a specific gene.

Allegedly the axis above gene zalpha11 ligand man localized near the gene of IL-2, which is located in the region of chromosome 4q, which, as shown, has a clutch with susceptibility to inflammatory disease of the digestive tract (IBD) including Crohn's disease (CD) and ulcerative colitis) in some families (Hampe et al., Am. J. Hum. Genet. 64:808-816, 1999; Cho et al., Proc. Natl. Acad. Sci. 95:7502-7507, 1998). In addition, gene zalpha11 receptor mapped relatively R, another genomic region, which is associated with susceptibility to Crohn's disease (CD) (Hugot et al., Nature 379:821-823, 1996; Ohmen et al., Hum. Mol. Genet. 5:1679-1683. 1996). Crohn's disease (CD) is a chronic inflammation of the digestive tract with frequent systemic involvement; although the exact etiology is unknown, immunoregulatory dysfunction, including lack of tolerance to normal antigens of the digestive tract, is the main component (for review see Braegger et al., Annals Allergy 72:135-141, 1994; Sartor, Am. J. Gastroenterol. 92:58-1 IS, 1997)). Several studies have found abnormal activity of MK-cells in patients with Crohn's disease (CD) (see, for example, Egawa et al., J. Clin. Lab. Immunol. 20:187-192. 1986; Aparicio-Pages et al., J. Clin. Lab. Immunol. 29:119-124, 1989; Tol et al., Scand. J. Gastroenterol. 27:999-1005, 1992)), and was also documented education In cells with defective memory (Brogan et al., J. Clin. Lab. Immunol. 24:69-74. 1987). Because zalpha11 ligand plays a role in immunoregulation and since g is HN a receptor, and ligand are in the areas of susceptibility to CD (Crohn's disease), as receptor and ligand are candidate genes for genetic predisposition to Crohn's disease.

The definition of participation zalpha 11-receptor and/or zalpha11 ligand in the pathology of IBD (inflammatory disease of the digestive tract) can be done in several ways. Sequencing of exons from genomic DNA may reveal coding mutations including missense mutations, nonsense mutations and mutation-shift frame (sign mutations)), and sequencing of cDNA. An additional advantage sequencing of genomic DNA lies in the fact that the boundaries of splicing are also contained in sequanorum fragments and can detect deviations splicing, which may not be present in the cDNA samples, if, for example, incorrectly splanirowannya RNA was quickly degradibility. Was determined the genomic structure of the zalpha11 ligand. Other methods of analysis zalpha 11-ligand and zalpha 11-receptor in patients with IBD include: (1) assessment of the production of the ligand of activated T-cells from patients compared with normal controls (i.e. using bioanalysis); (2) in situ hybridization of RNA zalpha 11-zalpha11 receptor or ligand with sections of inflamed intestine of IBD patients in comparison with similar slices from normal controls; (3) immunochemistry on Wed the Ah from IBD patients compared with normal controls; and (4) evaluation of autochemistry peripheral b cells to zalpha11 ligand as measured by analyses of mitogenesis.

Diagnostic agent could assist physicians in determining the type of disease and the appropriate associated therapy or could help in genetic counseling. Themselves antibodies against zalpha11 ligand, polynucleotide and polypeptides zalpha11 ligand of the present invention can be used for detection of the polypeptide, mRNA zalpha11 ligand or antibodies against zalpha11 ligand, serving thus as markers, and can be used directly for detection of genetic diseases or cancers, as described herein, using methods known in this field and are described here. Next, polynucleotide probes zalpha11 ligand can be used to detect abnormalities, including chromosome 4q27, as described here. These abnormalities may be associated with human diseases or oncogenesis, spontaneous miscarriage or other genetic disorders. Thus, the polynucleotide probes zalpha11 ligand can be used to detect abnormalities or genotypes associated with these defects.

As discussed above, defects in the gene zalpha11 ligand themselves can lead to hereditary pathological condition is found to be human. Molecules of the present invention, such as the polypeptides, antagonists, agonists, polynucleotides and antibodies of the present invention can contribute to the detection, diagnosis, prevention and treatment zabolevanii associated with a genetic defect zalpha11 ligand. In addition, polynucleotide probes zalpha11 ligand can be used to detect allelic differences between affected and not affected by the individuals in the chromosomal locus zalpha11 ligand. By themselves, the sequence zalpha11 ligand can be used as diagnostic agents in DNA profiling in forensic medicine.

In General, the diagnostic methods used in the analysis of genetic clutch for detecting genetic abnormalities or aberrations in the patient's well-known in this field. Most diagnostic methods include stage (i) obtaining a genetic sample from a potentially diseased patient, diseased patient or potential not diseased carrier of a recessive disease allele; (ii) to obtain the product of the first reaction by incubation of this genetic sample with a polynucleotide probe zalpha11 ligand, where this polynucleotide will gibridizatsiya with complementary polynucleotide sequence, for example, RFLP analysis, or in what ubytovanie this genetic samples from semantic and antimuslim primer in a PCR reaction under suitable conditions, the PCR reaction; (iii) visualization of the product of the first reaction by gel-electrophoresis and/or other known method, such as the visualization of the product of the first reaction polynucleotide probe zalpha11 ligand, where this polynucleotide will gibridizatsiya with the complementary polynucleotide sequence of the first reaction; and (iv) comparing the visualized first reaction product with the product of the second control reaction genetic samples from normal or control of the individual. The difference between the product of the first reaction and product control reaction is a sign of genetic abnormalities in ill or potentially ill patient or a sign of the presence of the phenotype of heterozygous recessive media metabolised patient or the presence of a genetic defect in a tumor from a diseased patient, or the presence of genetic abnormalities in the fetus or preimplantation embryo. For example, the difference in the distribution of restriction fragments, the length of PCR products, length of repetitive sequences in the genetic locus zalpha11 ligand, etc. is a sign of genetic abnormalities, genetic aberration, or allelic difference in comparison with normal controls. The controls can be from non-affected family members or unrelated individuals for whom threaded depending on the test and the availability of samples. Genetic samples for use in this invention include genomic DNA, mRNA and cDNA isolated from any tissue or other biological sample of the patient, such as, but not limited to, blood, saliva, semen, embryo cells, amniotic fluid, etc. Polynucleotide probe or primer can be RNA or DNA and a portion of SEQ ID NO:1, the complement SEQ ID NO:1 or its RNA equivalent. Such methods, showing the analysis of genetic clutch in relation to the phenotypes of human diseases, are well known in this field. In relation to methods based on PCR in the diagnosis, see, in General, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methods and Applications (Humana Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR (Humana Press, Inc. 1998) and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).

Mutations associated with a locus zalpha11 ligand can be detected using nucleic acid molecules of the present invention with a sheet of standard methods for direct mutation analysis, such as analysis of polymorphism of the lengths of restriction fragments, the analysis of short tandem repeats using PCR methods, analysis system, amplification-refractory (resistant) mutations, detection of polymorphism single-stranded conformation, ways Rasse the population using RNase, denaturing gradient gel electrophoresis, analysis of erroneous pairing using fluorescence and other genetic methods of analysis known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Humana Press, Inc. 1996), Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren (ed.), Laboratory Protocols for Mutation Detection (Oxford University Press 1996), Birren et al., (eds.), Genome Analysis, Vol. 2: Detecting Genes (Cold Spring Harbor Laboratory Press, 1998), Dracopoli et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, 1998) and Richards and Ward, "Molecular Diagnostic Testing," in Principles of Molecular Medicine, pages 83-88 (Humana Press, Inc. 1998). Direct analysis of gene zalpha11 ligand on the mutation can be performed using human genomic DNA. Methods amplification of genomic DNA, obtained, for example, from peripheral blood lymphocytes, known to experts in this field (see, for example, Dracopoli et al., (eds.), Current Protocols in Human Genetics, pages 7.1.6-7.1.7 (John Wiley & Sons 1998)).

The position of introns in the gene zalpha11 ligand was determined by identification of genomic clones and subsequent sequencing of the boundaries of the intron/exon. The first intron lies between amino acid residue 56 (Leu) and the residue 57 (Val) in SEQ ID NO:2 and has a length of 115 BP of the Second intron has a maximum length of 4.4 TPN and lies between amino acid residue 68 (Glu) and the remainder of 69 (Thr) in SEQ ID NO:2. Third intron has a length of 2.6 TPN and lies between amino acid the STATCOM 120 (Leu) and the residue 121 (Thr) in SEQ ID NO:2. End of the intron, 89 P.N., lies between amino acid residue 146 (Lys) and the remainder 147 (Met) in SEQ ID NO:2. Full gene extends over approximately 8 TPN

The structure of the gene zalpha11 ligand is similar to the structure of the gene IL-2 (Fujita et al., Proc. Natl. Acad. Sci. 80:7437-7441, 1983), although gene zalpha11 ligand contains one additional intron (intron 4). Character short of the first intron and a long second and third introns is conservative between these two genes, although the gene IL-2 is in General slightly smaller (about 6 TPN). Gene IL-15, on the other hand, consists of 8 exons and spans at least 34 TPN (Anderson et al. Genomics 25:701-706, 1995). Thus, gene zalpha11 ligand is more similar in structure with the gene of IL-2 than with the gene of IL-15.

In preferred embodiments of the present invention the selected encoding zalpha11 ligand molecules of nucleic acids will be gibridizatsiya under stringent conditions with nucleic acid molecules having the nucleotide sequence of SEQ ID NO:1, nucleic acid molecules having the nucleotide sequence of nucleotides 47-532 SEQ ID NO:1, or nucleic acid molecules Callot, complementary to SEQ ID NO:1. Typically, stringent conditions are chosen so that the temperature was about 5°With lower than point thermal melting point (Tmfor the specific sequence at opredelennyje strength and pH. Tmis the temperature (under defined ionic strength and pH)at which 50% of the target sequence hybridize with exactly compatible probe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA, can gibridizatsiya, if the nucleotide sequence have some degree of complementarity. Hybrids can tolerate incorrectly paired bases in the double helix, but the stability of the hybrid is affected by the degree of incorrect mating. Tmmistakenly paired hybrid is reduced by 1°for each 1-1,5% incorrect mating. Varying the stringency conditions of hybridization allows to control the degree of erroneous pairing, which will be present in this hybrid. The severity increases with the increase in the temperature of hybridization and the reduction of the ionic strength of the hybridization buffer.

The adaptation of these conditions for use with a specific polynucleotide hybrid is quite within the specialist qualification in this field. Tmfor a specific sequence of the target is the temperature (under certain conditions), at which 50% of the target sequence will be gibridizatsiya with just a compatible sequence of the probe. Conditions that affect Tminclude the size and content of par foundations of the deposits of this polynucleotide probe, ionic strength of the hybridization solution and the presence of destabilizing agents in the hybridization solution. Numerous equations for calculating the Tmknown in this field and are specific for DNA, RNA and hybrids, DNA-RNA, and polynucleotide sequences of the probes of varying length (see, for example, Sambrook et al., (Molecular Cloning. A Laboratory Manual, Second Edition Cold Spring Harbor Laboratory Press 1989); Ausubel et al., (eds.), Current Protocols in Molecular Biology (John Wiley and Sons, Inc., 1987); Berger and Kimmel (eds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev. Biochim. Mol. Biol. 26:227 (1990)). Software for sequence analysis, such as OLIGO 6.0 (LSR; Long Lake, MN) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, CA), as well as sites on the Internet are the available tools for the analysis of specific sequences and calculation of Tmbased on user-defined criteria. Such programs can also analyze the specific sequence under certain conditions and to identify the appropriate sequence of probes. Typically, hybridization of a longer polynucleotide sequences, >50 base pairs, performed at a temperature of approximately 20-25°calculated below Tm. For smaller probes, <50 base pairs, hybridization is usually carried out at Tmor 5-10°below. It allows to reach a maximum speed of hibri is Itachi for hybrids, DNA-DNA and DNA-RNA.

After hybridization of the nucleic acid molecule can be washed to remove not gibridizatsiya under strict conditions or under conditions of high stringency to the nucleic acid molecules. Typical stringent washing conditions include washing in a solution of 0.5x-2x SSC with 0.1% sodium dodecyl sulfate (LTOs) at 55-65°C. That is, the nucleic acid molecule encoding a variant zalpha11 polypeptide-ligand hybridize to a molecule of nucleic acid having the nucleotide sequence of SEQ ID NO:1 (or its complement)under stringent washing conditions, in which the stringency washing is equivalent to 0.5x-2x SSC with 0.1% LTOs at 55-65°including 0.5x SSC with 0,1% - ordinator at 55°With or 2x SSC with 0.1% LTOs at 65°C. the person skilled in the art will easily come up with equivalent conditions, for example, using replacement SSC on SSPE in the wash solution.

Typical conditions of high stringency washing include washing in a solution of 0,1-0,2x SSC with 0.1% sodium dodecyl sulfate (LTOs) at 50-65°C. in Other words, the molecules of nucleic acids encoding variant zalpha11 polypeptide-ligand hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 (or its complement)under conditions of high stringency washing, in which the severity of flushing equivalent to 0,1x-0,2x SSC with 0.1% LTOs at 50-65°With, including 0,1x SC with 0.1% LTOs at 50° With or 0,2x SSC with 0.1% LTOs at 65°C.

This invention also provides a dedicated zalpha11 polypeptide-ligand, which have essentially the same sequence identity with the polypeptide SEQ ID NO:2 or their orthologues. The term "essentially similar sequence identity" is used herein to denote polypeptides having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% identity to the sequences shown in SEQ ID NO:2, or their orthologues. The invention also includes polypeptides that contain amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity with amino acid residues 1-162 or 33-162 SEQ ID NO:2. The invention also includes molecules of nucleic acids that encode such polypeptides. Methods for determining percent identity are described below.

This invention also considers a variant nucleic acid molecule zalpha11 ligand, which can be identified using two criteria: a determination of the similarity between the encoded polypeptide with the amino acid sequence SEQ ID NO:2 and/or hybridization analysis described above. Such Varian is s zalpha11 ligand include molecules of nucleic acids: (1) that hybridize with a nucleic acid molecule, having the nucleotide sequence of SEQ ID NO:1 (or its complement) under stringent washing conditions, in which the stringency washing is equivalent to 0.5x-2x SSC with 0.1% LTOs at 55-65°With; or (2) that encode a polypeptide having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% identity with amino acid sequence SEQ ID NO:2. Alternative options zalpha11 ligand can be characterized by the nucleic acid molecules: (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 (or its complement) under the conditions of leaching high severity, in which the severity of flushing equivalent to 0,1x-0,2x SSC with 0.1% LTOs at 50-65°C; and (2) that encode a polypeptide having at least 70%, at least 80%, at least 90%, at least 95% or higher than 95% identity with amino acid sequence SEQ ID NO:2.

The percentage sequence identity define common ways. See, for example, Altschul et al., Bull. Math. Bio. 48:603 (1986) and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:of 10,915 (1992). Briefly, two amino acid sequences map building for optimization assessments mapping using opening penalty gap 10 and penalty extension gap 1 and the evaluation matrix "BLOSUM62" Henikoff and Henikoff (ibid.), a shown in table 4 (which minamikata presented using standard one-letter codes). Then the percentage identity is calculated as

The total number of identical matches ×100

[length of the longer sequence plus the number of gaps introduced into the longer sequence to map these two sequences]

Table 4
AndRNDQEGHILToMFPSTWYV
And4
R-15
N-206
D-2-216
0-3-3-39
Q-1100-3 5
E-1002-425
G0-20-1-3-2-26
H-201-1-300-28
I-1-3-3-3-1-3-3-4-34
L-1-2-3-4-1-2-3-4-324
To-120-1-311-2-1-3-25
M-1-1-2-3-10-2-3-212-15
F-2-3-3-3-2-3-3-3-100-306
P-1-2-2-1-3-1-1-2-2-3-3-1-2-47
S1-110-1000-1-2-20-1-2-14
T0-10-1-1-1-1-2-2-1-1-1-1 -2-115
W-3-3-4-4-2-2-3-2-2-3-2-3-11-4-3-211
Y-2-2-2-3-2-1-2-32-1-1-2-13-3-2-227
V0-3-3-3-1-2-2-3-331-21-1-2-20-3-14

Professionals in this field should be clear that there are many established algorithms for comparing two amino acid sequences. The search algorithm of similarity "FASTA" Pearson and Lipman before the hat is suitable for mapping proteins to study the level of identity, General described here for amino acid sequence and amino acid sequence of a hypothetical variant zalpha11 ligand. The algorithm FASTA described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), and Pearson, Meth. Enzvmol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifying regions shared in the requested sequence (for example, SEQ ID NO:2) and a test sequence that have either the highest density of identities (if the ktup variable is 1)or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions or deletions. Then the ten areas with the highest density of identities re-evaluate by comparing the similarities of all pairs of amino acids using a matrix of amino acid substitutions, and the ends of the districts "straighten" to include only residues that contribute the highest evaluation. If you have multiple areas with grades greater than the value of the "cut-off" (calculated by a predetermined formula based on the length of the sequence and its value ktup), then the order given in the original trim areas are examined to determine whether or not these areas are to be connected with the formation of approximate matching with gaps. Finally, the areas with the highest rating of two amino acid sequence is of linesta map using a modification of the algorithm of Needleman-Wunsch-Sellers (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which makes possible insertions and deletions of amino acids. The preferred parameters for FASTA analysis are: ktup=1, opening penalty gap=10, penalty extension gap=1 and the matrix of substitution=LOSUM62. These parameters can be entered into the program FASTA-file modification evaluation matrix ("SMATRIX"), as explained in Appendix 2 Pearson, Meth. Enzymol. 183:63 (1990).

The FASTA program can also be used to determine the identity of the molecules of nucleic acids using the above ratio. For comparisons of nucleotide sequences of the ktup value can be between 1 and 6, preferably from 3 to 6, most preferably 3, with other parameters set as default.

Variant polypeptides zalpha11 ligand with essentially the same sequence identity are characterized by the fact that they have one or more amino acid substitutions, deletions or accessions. These changes are preferably are of a minor nature, that is conservative replacement amino acids (see table 5) and other substitutions that do not affect significantly to the installation or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino - or carboxyl-terminal extension, such as aminobenzoic a methionine residue, a small linker peptide is the ID of up to about 20-25 residues, or affinity label. Thus, the invention includes polypeptides of from about 108 to about 216 amino acid residues that comprise a sequence that is at least 70%, preferably 90% and more preferably 95% or more identical to the corresponding region SEQ ID NO:2. Polypeptides containing affinity tags can further comprise the site of proteolytic cleavage between the zalpha11 polypeptide-ligand and this affinity tag. Suitable sites include the sites of cleavage by thrombin and the sites of cleavage by factor XA.

Table 5
The conservative replacement amino acids
Main:arginine

lysine
histidine
Sour:glutamic acid
aspartic acid
Polar:glutamine
asparagine
Hydrophobic:leucine
isoleucine
valine
Aroma: phenylalanine
tryptophan
tyrosine
Small:glycine
alanine
series
threonine
methionine

Can be performed identify amino acid residues that contain regions or domains that are critical for the preservation of structural integrity. In these areas it is possible to identify specific residues, which will be more or less resistant to change or to keep the overall tertiary structure of this molecule. Methods of analysis of the structure of the sequence include, but are not limited to, a comparison of multiple sequences with high identity with amino acids or nucleotides, the tendency to form secondary structures, binary distribution, complementary packing and hidden polar interactions (Barton, Current Qpin. Struct. Biol. 5:372-376, 1995 and Cordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). Usually when designing modifications to molecules or to identify specific fragments of the structure definition will be accompanied by an estimation of activity of modified molecules.

Changes in amino acid sequence you anaut in the zalpha11 polypeptide-ligand thus, to minimize the destruction of the structure of a higher order, which are essential for biological activity. For example, when the zalpha11 polypeptide-ligand contains one or more helices, changes in amino acid residues will be carried out in such a way as not to interfere with the geometry of the coils and other components of the molecule where changes in conformation reduce some critical function, for example, the binding of this molecule to its binding partners, for example, helices a and D residues 44, 47 and 135 of SEQ ID NO:2. The effects of changes in amino acid sequence can be predicted, for example, computer modeling, as described above, or determined by analysis of crystal structure (see, for example, Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995). Other methods that are well known in the field, compare the packing variant protein with a standard molecule (e.g., the native protein). For example, can be made by comparing the distribution of cysteine in variance and standard molecules. Mass spectrometry and chemical modification using recovery and alkylation provide methods for determination of cysteine residues that are associated with disulfide bonds or free from such associations (Bean et al., Anal. Biochem. 201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993, and Patterson et al., Anal. Chem. 66:3727-3732, 1994). About the commonly considered what if the modified molecule does not have the same distribution of cysteine as standard molecule, stacking may be compromised. Another well-known and accepted method of measuring styling is a circular dichroism (CD). The measurement and comparison of the CD spectra generated by a modified molecule and the standard molecule is routine (Johnson, Proteins 7:205-214. 1990). Crystallography is another well-known method for the analysis of styling and patterns. Nuclear magnetic resonance (NMR), enzymatic peptide mapping and mapping of epitopes are also known methods for the analysis of similarity styling and patterns between proteins and polypeptides (Schaanan et al., Science 257:961-964, 1992).

Can be obtained profile hydrophilicity Hopp/Woods protein sequence zalpha11 ligand shown in SEQ ID NO:2 (Norr et al., The OEWG. Natl. Acad. Sci. 78:3824-3828, 1981; Norr, J. Immun. Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering 11:153-169, 1998). This profile is based on a sliding window of six residues. Hidden remains of G, S and T and exposed (open) residues of N, Y and W are not taken into account. For example, in zalpha11-ligand hydrophilic regions include amino acid residues 114-119 SEQ ID NO:2, amino acid residues 101-105 SEQ ID NO:2, amino acid residues 126-131 SEQ ID NO:2, amino acid residues 113-118 SEQ ID NO:2 and amino acid residues 158-162 SEQ ID NO:2.

Specialists in this field will be clear that the hydrophilicity or hydrophobicity should be taken into account when designing modifications in the amino acid sequence of the polypeptide zalpha11 ligand, so as not to disrupt the overall structural and biological profile. Of particular interest to replace represent hydrophobic residues selected from the group consisting of Val, Leu, and Not, or from the group consisting of Met, Gly, Ser, Ala, Tyr and Trp. For example, residues that are resistant to replacement could include residues 100 and 103, shown in SEQ ID NO:2. Residues of cysteine at positions 71, 78, 122 and 125 of SEQ ID NO:2 will be relatively unstable to be replaced.

The identities of essential amino acids can also be inferred from analysis of sequence similarity between IL-15, IL-2, IL-4 and GM-CSF with zalpha11-ligand. Using methods such as analysis of "FASTA", described above, regions of high similarity identify the family of proteins and is used for the analysis of amino acid sequences for conservative districts. An alternative approach to the identification of variant polynucleotide zalpha11 ligand on the basis of structure is to determine whether a nucleic acid molecule encoding a potential gene variant zalpha11 ligand, gibridizatsiya with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1, as discussed above

Other methods of identification of essential amino acids in the polypeptides of this invention are known in this field, such as site-directed mutagenesis or Leninskoye mutagenesis (Cunningham and Wells, Science 244:1081 (1989); Bass et al., Proc. Natl. Acad. Sci. USA 88:4498 (1991), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering" in Proteins: Analysis and Design. Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In the latter method, single alanine mutations introduced at every residue in the molecule, and the resulting mutant molecules are tested for biological or biochemical activity as described below, to identify amino acid residues that are critical for activity of this molecule. Cm. also Hilton et al., J. Biol. Chem. 271:4699 (1996).

This invention relates also to the functional fragments of the polypeptides zalpha11 ligand and the molecules of nucleic acids encoding such functional fragments. "Functional" zalpha11 ligand or a fragment, defined here differs by its proliferative or differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to specifically bind with an antibody against zalpha11 ligand or receptor zalpha11 ligand (soluble or immobilized). As described previously, zalpha11 ligand has the structure h is directparallel beam, contains helix (amino acid residues 41-56), helix (amino acid residues 69-84), helix (amino acid residues 92-105) and helix D amino acid residues 135-148), as shown in SEQ ID NO:2. Thus, the invention additionally provides fused (hybrid) protein, comprising: (a) polypeptide molecules containing one or more spirals described herein; and (b) functional fragments containing one or more of these spirals. The other polypeptide portion of the fused protein can consist of another consisting of chetyrekhmernogo bundle cytokine, such as IL-15, IL-2, IL-4 and GM-CSF, or unnatural and/or an unrelated secretory signal peptide that facilitates secretion of the fused protein.

Thus, this invention provides fused proteins containing at least four polypeptide, where the order of polypeptides from N-Terminus to the C-end is as follows: first, the polypeptide contains amino acids selected from the group consisting of: (a) amino acid residues 36-46 SEQ ID NO:111 helix A of IL-2; (b) amino acid residues 29-43 SEQ ID NO:112 spiral And IL-15; (C) amino acid residues 45-68 SEQ ID NO:113 helix A of IL-4; (d) amino acid residues 30-44 SEQ ID NO:114 spiral And GM-CSF, and (e) amino acid residues 41-56 SEQ ID NO:2; a first spacer of 6-27 amino acids; and a second polypeptide that contains amino acid mod and, selected from the group consisting of: (a) amino acid residues 53-75 SEQ ID NO:111 spiral In IL-2; (b) amino acid residues 65-83 SEQ ID NO:112 spiral In IL-4; (C) amino acid residues 84-101 SEQ ID NO:113 spiral In IL-15; (d) amino acid residues 72-81 SEQ ID NO:114 spiral In GM-CSF and (e) amino acid residues 69-84 SEQ ID NO:2; a second spacer of 5-11 amino acid residues; a third polypeptide that contains amino acid residues selected from the group consisting of (a) amino acid residues 87-99 SEQ ID NO:111 spiral With IL-2; (b) amino acid residues 95-118 SEQ ID NO:112 spiral With IL-4; (C) amino acid residues 107-119 SEQ ID NO:113 spiral With IL-15; (d) amino acid residues 91-102 SEQ ID NO:114 spiral With GM-CSF and (e) amino acid residues 92-105 SEQ ID NO:2; a third spacer of 3-29 amino acid residues; and a fourth polypeptide that contains amino acid residues selected from the group consisting of (a) amino acid residues 103-121 SEQ ID NO:111 helix D of IL-2; (b) amino acid residues 134-157 SEQ ID NO:112 helix D of IL-15; (C) amino acid residues 134-160 SEQ ID NO:113 helix D of IL-4; (d) amino acid residues 120-131 SEQ ID NO:114 helix D of GM-CSF and (e) amino acid residues 135-148 SEQ ID NO:2; where at least one of the four polypeptides is a zalpha1 1-ligand. In other embodiments, spacer elements peptides will be selected from loops a/b and C/D zalpha11 ligand, IL-2, IL-4, IL-15 or GM-CSF, as shown in table 1.

Routine deletion analysis the s molecules of nucleic acids can be performed to obtain functional fragments of a nucleic acid molecule, encodes a polypeptide zalpha11 ligand. As an illustration, DNA molecules having the nucleotide sequence of SEQ ID NO:1, or fragments thereof, can be cleaved by the nuclease l31 obtaining a number of "embedded" deletions. Then these DNA fragments insertyour in expressing vectors in proper reading frame, and the expressed polypeptides isolated and tested for the activity of zalpha11 ligand, or the ability to bind with antibodies against zalpha11 ligand or receptor zalpha1 1-ligand. One alternative to splitting ectonucleoside is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired fragment of the zalpha11 ligand. Alternatively, fragments of the gene zalpha11 ligand can be synthesized using the polymerase chain reaction.

Standard methods of identification of functional domains are well known to persons of ordinary skill in this field. For example, research on one or both ends of the truncated interferons summarized Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995). In addition, standard methods for the functional analysis of proteins is described, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993); Content et al., "Expression and preliminary deletion analysis of the 42 kDa 2-5A synthetase induced by human interferon," in Biological Interferon Systems. Proceedings of ISIR-TNO Meeting o Interferon Systems. Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, "The EGF Receptor," in Control of Animal Cell Proliferation 1, Boynton et al., (eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol. Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995) and Meisel et al., Plant Molec. Biol. 30:1 (1996).

Numerous amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as described Reidhaar-Olson and Sauer (Science 241:53. 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152, 1989). Briefly, these authors describe how simultaneous randomization of two or more positions in the polypeptide of selection on functional polypeptide and then sequencing mutagenically polypeptides to determine the range of allowable substitutions at each position. Other methods that may be used include phage way of presentation, e.g., Lowman et al., Biochem. 30:10832, 1991; Ladner et al., U.S. Patent No. 5223409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA7:127, 1988).

Options described nucleotide and polypeptide sequences zalpha11 ligand can be formed by DNA shuffling, described by Stemmer, Nature 370:389, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747, (1994) and in international publication WO 97/20078. Briefly, variant DNA molecules generated through homologous recombination in vitro random fragmentation of the original DNA with subsequent re-sat is rcoi using PCR, leading to accidentally introduced tockovi mutations. This method can be modified using the family of the original DNA molecules, such as allelic variants or DNA molecules from different species, to introduce additional variability into the process. Selection or screening for the desired activity, and then further repetitions of mutagenesis and analysis provides rapid "evolution" of the sequences by selection for the desired mutation with simultaneous selection against harmful changes.

Methods of mutagenesis, described above, can be combined with high-performance, automated screening methods to detect activity of cloned mutagenically polypeptides in the cells of the host. Methanediamine DNA molecules that encode biologically active polypeptides, or polypeptides that can be contacted with antibodies against zalpha11 ligand, or soluble zalpha11 receptor, can be extracted from the host cells and rapidly sequenced using modern equipment. These methods make it possible to quickly determine the importance of individual amino acid residues in the interest of the polypeptide and can be applied to polypeptides of unknown structure.

In addition, proteins of this invention (or their polypep the derivative fragments) can be combined with other bioactive molecules, in particular, other cytokines, to ensure polyfunctional molecules. For example, one or more spirals of zalpha11 ligand may be linked with other cytokines to enhance the biological properties or efficiency gain.

Thus, this invention provides a number of new hybrid molecules in which a segment containing one or more spirals zalpha11 ligand, fused with another polypeptide. The fusion preferably is produced by splicing at the DNA level to make possible the expression of chimeric molecules in recombinant systems receive. The resulting molecules are then analyze properties such as improved solubility, improved stability, prolonged half-life clearance, improved levels of expression and secretion and pharmacodynamics. Such a hybrid molecule may also contain additional amino acid residues (e.g., a polypeptide linker) between the components-proteins or components polypeptides.

Not occurring natural amino acids include, without limitation, TRANS-3-methylpropyl, 2,4-methanoproline, CIS-4-hydroxyproline, TRANS-4-hydroxyproline, N-methylglycine, ALLO-threonine, methylthionine, hydroxyethylation, hydroxyethylrutoside, nitroglycerin, hemaglutinin, pipecolinic acid, thiazolidinediones sour is, digitopolis, 3 - and 4-methylpropan, 3,3-dimethylpropyl, tert-leucine, Norvaline, 2-aseptically, 3-aseptically, 4-aseptically and 4-forfinally. In this area there are several ways to include not natural occurring amino acid residues in proteins. For example, can be used in vitro system, in which nonsense mutations are suppressed with the use of chemically aminoacylating suppressor tRNA. Methods of synthesis of amino acids and tRNA aminoacylation known in this field. Transcription and translation of plasmids containing nonsense mutations, usually in a cell-free system containing an extract of the E. coli S30 and commercially available enzymes and other reagents. Proteins purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722. 1991; Ellman et al., Methods Enzvmol. 202:301, 1991; Chung et al.. Science 259:806. 1993 and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145. 1993).

In the second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylating suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991, 1996). In the third method, the E. coli cells cultivated in the absence of natural amino acids to be substituted (e.g., phenylalanine), and in the presence of desirable not natural occurring amino acids (amino acids) (e.g., 2-azaphenanthrene, 3-azaphenanthrene, 4-azaphenanthrene or 4-pertanyaannya. Natural is not occurring amino acid is incorporated into the protein instead of its natural copy. Cm. Koide et al., Biochem. 33:7470, 1994. The naturally occurring amino acid residues can be converted into natural not found varieties of chemical modification in vitro. Chemical modification can be combined with site-directed mutagenesis to further extend the range of substitutions (Wynn and Richards, Protein Sci. 2:395. 1993).

It may be useful to stabilize zalpha11 ligand to extend the period of existence of this molecule, in particular, to extend metabolic persistence in an active state. To achieve the extended half-period of the existence of molecules zalpha11 ligand can be chemically modified using the methods described here. Pegylation is one of the methods commonly used, which showed an increase of half existence in plasma, increased solubility and reduced antigenicity and immunogenicity (Nucci et al., Advanced Drug Delivery Reviews 6:133-155, 1991, and Lu et al. Int. J. Peptide Protein Res. 43:127-138, 1994).

Amino acid residues zalpha11 ligand can be replaced by a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, which is not found in natural amino acids and unnatural amino acids.

This invention also provides the polyp is tianye fragments or peptides, containing the epitope-bearing portion of the zalpha11 polypeptide-ligand described herein. Such fragments or peptides may contain an "immunogenic epitope", which is part of the protein, which causes the humoral response (antitelomerase) when using the whole protein as immunogen. Immunogenic epitope-bearing peptides can be identified using standard methods (see, e.g., Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998 (1998)).

In contrast, polypeptide fragments or peptides may contain "antigenic epitope", which is a region of a protein molecule that can specifically bind the antibody. Certain epitopes consist of linear or continuous segment of amino acids, and the antigenicity of such epitope is not destroyed denaturing agents. In this area it is known that relatively short synthetic peptides that mimic epitopes of the protein, can be used to stimulate the formation of antibodies against this protein (see, e.g., Sutcliffe et al., Science 219:660 (1983)). Thus, antigenic epitope-bearing peptides and polypeptides of the present invention is applicable for the induction of antibodies that bind to polypeptides described herein. The hydrophilicity profiles Norr-Woods can be used to identify areas that have the most activisation potential (Norr et al., 1981, ibid and Norr, 1986, ibid.). In zalpha11-ligand these regions include amino acid residues 114-119, 101-105, 126-131, 113-118 and 158-162 SEQ ID NO:2.

Antigenic epitope-bearing peptides and polypeptides preferably contain at least four to ten amino acids, at least ten to fourteen amino acids, or about fourteen to about thirty amino acids of SEQ ID NO:2 or SEQ ID NO:56. Such epitope-bearing peptides and polypeptides can be obtained by fragmenting the polypeptide zalpha11 ligand or by chemical peptide synthesis, as described herein. In addition, the epitopes can be selected using phage representation libraries of random peptides (see, for example, Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993) and Cortese et al., Curr. Qpin. Biotechnol. 7:616 (1996)). Standard ways of identifying epitopes and receive antibodies from small peptides that contain the epitope described, for example, the Mole, "an epitope Mapping," in Methods in Molecular Biology. Vol. 10, Manson (ed.), pages 105-116 (The Humana Press, Inc. 1992); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies: Production. Engineering and Clinical Application, Ritter and Ladyman (eds.), Pages 60-84 (Cambridge University Press 1995), and Coligan et al., (eds.), Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons 1997).

Regardless of the particular nucleotide sequence of the variant polynucleotide zalpha11 ligand this polynucleotide encodes a polypeptide that is characterized by its proliferative or differencer the overall activity, its ability to induce or inhibit specialized cell function or the ability to specifically bind with an antibody against zalpha11 ligand or zalpha11 receptor. More specifically, variant polynucleotide zalpha11 ligand will encode polypeptides which exhibit at least 50% and preferably more than 70, 80 or 90% of the activity of the polypeptide shown in SEQ ID NO:2.

For any of the zalpha11 polypeptide-ligand, including variants and fused proteins, the person skilled in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information presented in tables 1 and 2 above.

Further, this invention provides various other polypeptide fusion (and related multimeric proteins containing one or more fused polypeptides). For example, the zalpha11 polypeptide-ligand can be obtained in the form of a merger with dimerized protein, as described in U.S. patent numbers 5155027 and 5567584. Preferred dimerized proteins in this regard include the domains of the constant regions of immunoglobulins. The polypeptide of the fusion immunoglobulin-zalh11-ligand can be expressed in genetically engineered cells to produce different multimeric analogues zalpha11 ligand). Auxiliary domain may be fused with polypeptide zalpha11 ligand to target them to specific cells, tissues, or macromolecules. For example, the polypeptide or protein zalpha11 ligand could be aimed at a given cell type merge zalpha11 polypeptide-ligand with a ligand that specifically binds to a receptor on the surface of the target cells. In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. The zalpha11 polypeptide-ligand may be fused to two or more parts of the molecule, such as affinity tag for purification and a targeting domain. Polypeptide fusion may also contain one or more cleavage sites, in particular, between domains. Cm. Tuan et al., Connective Tissue Research 34:1-9, 1996.

Using the methods discussed here specialist in this area may identify and/or receive a different polypeptides, which have essentially the same sequence identity with residues 1-162 or 33-162 SEQ ID NO:2, or their functional fragments and merge, where such polypeptides or fragments retain the properties of the wild-type protein, such as the ability to stimulate proliferation, differentiation, induce specialized cellular function or link zalpha11 receptor or antibodies against zalpha11 ligand.

Polypeptides zalpha11 ligand of the present invention, including full-sized polypeptides, functional fragments and hybrids the e polypeptides, can be produced in genetically engineered cells-owners in accordance with conventional ways. Appropriate cell hosts are those the types of cells that can be transformed or transliterowany exogenous DNA and grown in culture, and they include bacteria, fungal cells and cultivated in higher eukaryotic cells. Eukaryotic cells, in particular, cultivated cells of multicellular organisms are preferred. Ways of manipulating cloned DNA molecules and introducing exogenous DNA into a variety of cell hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. (2nd ed.) (Cold Sprinq Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 and Ausubel et al. (eds.) Current Protocols in Molecular Biology. John Wiley and Sons, Inc., NY, 1987.

In General, the DNA sequence encoding the polypeptide zalpha11 ligand, functionally linked to other genetic elements required for its expression, generally including the promoter and terminator of transcription in expressing vector. This vector will usually also contain one or more breeding markers and one or more points of initiation of replication, although experts in the field will be clear that in some systems of breeding markers may be provided on separate vectors and replication of exogenous DNA can be the ü provided by integration into the genome of the host cell. The choice of promoters, terminators, breeding markers, vectors and other elements is a matter of routine design within the ordinary skill in this field. Many of these elements are described in the literature and are available through commercial suppliers.

For the direction of the zalpha11 polypeptide-ligand in the secretory path of the host cell in expressing vector provide a secretory signal sequence (also known as a leader sequence, a pre-proposedvalue or pre sequence). The secretory signal sequence may be a signal sequence zalpha11 ligand or may be derived from another secreted protein (e.g., t-PA (tissue plasminogen activator)or synthesized de novo. The secretory signal sequence is functionally linked to a DNA sequence zalpha11 ligand, i.e. these two sequences are joined in the correct reading frame and is correctly placed for the direction of the newly synthesized polypeptide into the secretory path of the host cell. Secretory signal sequences are typically located 5' (left) from the DNA sequence that encodes an polypeptide, although certain signal sequences can be located in another month the e in the interest of the DNA sequence (see, for example, Welch et al., U.S. Patent No. 5037743; Holland et al., U.S. Patent No. 5143830).

Alternatively, a secretory signal sequence contained in the polypeptides of the present invention, is used to direct other polypeptides into the secretory pathway. This invention provides such a hybrid (merged) polypeptides. Can be made of the signal hybrid polypeptide in which a secretory signal sequence derived from amino acid residues 1-31 SEQ ID NO:2, functionally linked to a DNA sequence, encoding another polypeptide using methods known in this field and are described here. The secretory signal sequence contained in the hybrid polypeptides of the present invention, preferably fused at the amino-end with additional peptide for the direction of this additional peptide into the secretory pathway. Such structures have numerous applications, known in this area. For example, these new hybrid design with a secretory signal sequence can direct the secretion of the active component is not normally secreted protein. Such hybrid proteins can be used in vivo and in vitro for the direction of peptides through the secretory pathway.

Cultured mammalian cells are suitable hosts in this invented the I. Methods of introducing exogenous DNA into cells-mammalian hosts include mediated by calcium phosphate transfection (Wigler et al., Cell.14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), mediated DEAE-dextran transfection (Ausubel et at., ibid.) and mediated by liposomes transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993) and viral vectors (Miller and Rosman, BioTechnigues 7:980-90, 1989; Wang and Finer, Nature Med. 2:714-6, 1996). The preparation of recombinant polypeptides in cultured mammalian cells are described, for example, Levinson et al., U.S. Patent No. 4713339; Hagen et al., U.S. Patent No. 4784950; Palmiter et al., U.S. Patent No. 4579821; and Ringold, U.S. Patent No. 4656134. Suitable kulturama mammalian cells include cell lines COS-1 (ATS No. CRL 1650), COS-7 (ATSS No. CRL 1651), KSS (ATSS No. CRL 1632), KSS 570 (ATSS No. CRL 10314), 293 (ATSS No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72 (1977) and cell line of Chinese hamster ovary (e.g., Cho-K1; ATS No. CCL 61). Additional suitable cell lines are known in this field and available from public depositories such as the American type culture collection (American Type Culture Collection, Manassas, VA). In General, preferred strong promoters of transcription, such as promoters from SV-40 or cytomegalovirus. See, for example, U.S. Patent No. 4956288. Other suitable promoters include the promoters from the genes of metallothionein (U.S. atent No. 4579821 and U.S. Patent No. 4601978) and major late promoter of adenovirus.

The selection of the drug is usually used for screening in cultured mammalian cells, in which was embedded with alien DNA. These cells commonly called "the transfectants". Cells that were cultured in the presence of the selective agent and is able to convey the interest of the gene to their offspring, called "stable transfectants". Preferred breeding marker is a gene encoding resistance to the antibiotic neomycin. The selection is carried out in the presence of drug type neomycin, such as G-418, or other selection Systems can be used to increase the level of expression of the gene of interest, method, called "amplification". Amplification spend the cultivation of transfectants in the presence of low levels of the selective agent and then increase the amount of selective agent for selection of cells that produce high levels of products introduced genes. Preferred amplificare breeding marker is dihydrotetrazolo that tells the cells resistant to methotrexate. Other genes of drug resistance (e.g. resistance to hygromycin, multiple drug resistance,parameterizedthreadstart) can also be used. Alternative markers that introduce a modified phenotype, such as green fluorescent protein or cell surface proteins such as CD4, CD8, MHC Class I, alkaline phosphatase placenta, can be used for sorting of transfected cells from nitrostilbene cells by means of FACS sorting (sorting of cells with fluorescence excitation) or the method of separation using magnetic beads.

Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and cells of the birds. The use of Agrobacterium rhizogenes as a vector for gene expression in plant cells is considered Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation of insect cells and getting them alien polypeptides described Guarino et al., U.S. Patent No. 5162222 and WIPO publication WO 94/06463. Based on insect cells can be infected with recombinant baculovirus, usually produced from nuclear polyhedrosis virus of Autographa califomica (AcNPV). Cm. King, L.A. and R.D. Possee, The Baculovirus Expression System: A Laboratory Guide. London, Chapman and Hall, D.R. O'reilly et al., Baculovirus Expression Vectors: A Laboratory Manual. New York, Oxford University Press., 1994; and Richardson, C.D., Ed., Baculovirus Expression Protocols. Methods in Molecular Bioloav. Totowa, NJ, Humana Press, 1995. The second method of preparation of recombinant baculovirus using the system-based transposon described by Luckow (Luckow et al., J. Virol. 67:4566-79, 1993). This system produets is in the set of Bac-to-You™ (Life Technologies, Rockville, MD). This system uses vector-vector, pFastBac1™ (Life Technologies)containing transposon TP, to move the DNA encoding the polypeptide zalpha11 ligand, in a baculovirus genome maintained in E. coli as a large plasmid called a "bacmids". Vector-vector pFastBac1™ uses the promoter AcNPV polyhedrin to run the expression of gene of interest, in this case, the zalpha11 ligand. However, pFastBac1™ can be modified to a considerable extent. The polyhedrin promoter may be deleted and replaced by the promoter of the main protein of baculovirus (also known as Rag, R or promoter Mr), which is expressed early in infection with baculovirus, and it was shown that it is preferable for expression of secreted proteins. Cm. Hill-Perkins, M.S. and Possee, R.D. J. Gen. Virol. 71:971-6. 1990; Bonning, B.C. et al., J Gen. Virol. 75:1551-6, 1994 and Chazenbalk, G.D. and Rapoport, B. J. Biol. Chem. 270:1543-9, 1995. In such constructions vectors vectors can be used short or long version of the main protein promoter. In addition, can be constructed vectors-vectors that replace secretory signal sequences native zalpha11 ligand secretory signal sequences derived from proteins of insects. For example, a secretory signal sequence from ecdysteroidogenesis (EG), Melitina honey bees (Invitrogen, Carlsbad, CA) or Dr baculovirus (PharMingen, San Diego, CA) can be used in constructs to replace the secretion signal sequence of native zalpha11 ligand. In addition, vectors-vectors can contain merge in reading frame with DNA encoding an epitope tag at the C - or N-end expressed polypeptide zalpha11 ligand, such as an epitope tag, Glu-Glu (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using a method known in this field, vector-vector containing zalpha11 ligand, transformed in E. coli and subjected to screening for bacmid, which contain an interrupted lacZ gene indicative of recombinant baculovirus. Backmenu DNA containing the recombinant baculovirus genome, isolated by known methods and used for transfection of cells Spodoptera frugiperda, such as Sf9 cells. Then get the recombinant virus expressing zalpha11 ligand. Source materials recombinant virus prepared by the methods usually used in this field.

This recombinant virus is used to infect host cells, usually cell lines derived from autumn "field (military) worms, Spodoptera frugiperda. See, in General, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA. ASM Press, Washington, D.C., 1994. Other suitable cell line is kletos the th line of High FiveO™ (Invitrogen), produced from Trichoplusia ni (U.S. Patent No. 5300435).

Fungal cells, including yeast cells, can also be used in this invention. The species of yeast, of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastohs and Pichia methanolica. Methods of transformation of S. cerevisiae cells exogenous DNA and obtaining from them the recombinant polypeptides are described, for example, Kawasaki, U.S. Patent No. 4599311; Kawasaki et al., U.S. Patent No. 4931373; Brake, U.S. Patent No. 4870008; Welch et al., U.S. Patent No. 5037743 and Murray et al., U.S. Patent No. 4845075. Transformed cells are selected based on phenotype, defined breeding marker, usually in terms of resistance to drug or ability to grow in the absence of specific nutrients (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is a vector system NOT described by Kawasaki et al. (U.S. Patent No. 4931373), which allows to select the transformed cells on growth in containing glucose environment. Suitable promoters and terminators for use in yeast include promoters and terminators of the genes of the glycolytic enzymes (see, for example, Kawasaki, U.S. Patent No. 4599311; Kingsman et al., U.S. Patent No. 4615974 and Bitter, U.S. Patent No. 4977092) and alcohol dehydrogenase genes. Cm. also U.S. Patent No. 4990446, 5063154, 5139936 and 4661454. System transformation for other yeasts, including Hansenula polymorpha, Scizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillennondii and Candida maltosa, known in this area. See, for example, Gleeson et al., J. Gen. Environ. 132:3459-65, 1986 and Cregg, U.S. Patent No. 4882279. Cells Aspergitlus can be used in accordance with the methods McKnight et al., U.S. Patent No. 4935349. Methods of transformation of Acremonium chrysogenum described Sumino et at., U.S. Patent No. 5162228. Methods of transformation of Neurospora described Lambowitz, U.S. Patent No. 4486533.

The use of Pichia methanolica as a host for production of recombinant proteins is described in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536 and WO 98/02565. DNA molecules for use in transformation of P. methanolica are usually obtained in the form of a double-stranded circular plasmid, which preferably linearized before transformation. To obtain polypeptides in P. methanolica preferably, the promoter and terminator in this plasmid were the promoter and the terminator of the gene P. methanolica, such as gene utilization of alcohol P. methanolica (AUG1 or AUG2). Other applicable promoters include promoters of genes dihydroxyacetophenone (DHAS), formiatehydrogenlyase (FMD) and catalase (CAT). To facilitate integration of this DNA into the chromosome of the host, it is preferable to have the entire segment of the expression of this plasmid is flanked on both ends by DNA sequences of the host. Preferred breeding marker for use in Pichia methanolica is the ADE2 gene P. methanolica, which encodes FOS is ribosyl-5-iminoimidazolidine (AIRC; EC 4.1.1.21), which allows the cells to the owners ade2 to grow in the absence of adenine. For large-scale industrial processes where it is desirable to minimize the use of methanol, it is preferable to use cells of the host, in which both delegated gene utilization of methanol (AUG1 and AUG2). To obtain secreted proteins preferred cell owners defective in genes using proteases (RER and PRB1). Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding interest polypeptide, in cells of P. methanolica. It is preferable to transform cells of P. methanolica by electroporation using an exponentially decaying pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and the time constant ( Ω) from 1 to 40 milliseconds, most preferably about 20 milliseconds.

Prokaryotic cells-owners, including strains of bacteria Escheiichia coli, Bacillus and other genera are also applicable cells masters in this invention. Ways of transforming these hosts and expression of cloned them alien DNA are well known in the art (see, for example, Sambrook et al., ibid.). When the expression of zalpha11 polypeptide-ligand in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically in the IDA insoluble granules, or may be directed to periplasmatic space of a bacterial secretion sequence. In the first case, these cells are lysed and the granules are recovered and denatured using, for example, guanidinosuccinic or urea. Then denatured polypeptide can be re-laid and dimerizer by diluting the product denaturation, for example, by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against buffered saline. In the latter case, the polypeptide can be extracted from periplasmatic space in a soluble and functional form by the destruction of these cells (for example, by sonication or osmotic shock) to release content periplasmatic space and extract this protein, whereby eliminating the need for denaturation and re-installation.

Transformed and transfetsirovannyh cell hosts are cultivated in accordance with conventional procedures in a culture medium containing nutrients and other components required for growth of the selected host cells. Various suitable environment, including the environment with a certain composition of the medium and complex environment, known in this area and usually include the source of the IR carbon the nitrogen source, essential amino acids, vitamins and mineral compounds. The medium may also contain components such as growth factors or serum if required. The growing medium is usually to make a selection for cells containing exogenously added DNA, for example through selection using drugs or inadequacy of essential nutritional component, which is complemented by a breeding marker, available at expressing vector or cotransfected cells of the host. Cells of P. methanolica is cultivated in a medium containing adequate sources of carbon, nitrogen and trace elements, at a temperature of approximately 25-35°C. Liquid cultures provide sufficient aeration using conventional means, such as shaking a small flasks or bubbling fermentors. The preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Bacto™ Peptone (Difco Laboratories, Detroit, Ml), 1% Bacto™ yeast extract (Difco Laboratories), of 0.004% adenine and 0.006% L-leucine).

It is preferable to purify the polypeptides of the present invention to ≥80% purity, more preferably to ≥90% purity, even more preferably up to ≥95% purity, and particularly preferably up to pharmacologically pure state, i.e. to purity more than 99.9%, in relation to pollute their macromolecules, particularly other proteins and nucleic acids, and infectious and pyrogenic agents. Preferably, the purified polypeptide is essentially not containing other polypeptides, particularly other proteins of animal origin.

Expressed recombinant polypeptides zalpha11 ligand (or chimeric polypeptides zalpha11 ligand) can be purified using fractionation and/or conventional methods and environments clean. For fractionation of samples can be used for the deposition of ammonium sulfate and acid or jatropha extraction. Examples of stages of treatment may include the use of hydroxyapatite, gel-filtration, liquid Express chromatography of proteins (FPLC) and high performance liquid chromatography with reversed phase. Suitable chromatographic medium include derivatives of dextran, agarose, cellulose, polyacrylamide, special silica, etc. are Preferred derivatives of PEI, DEAE, QAE and Q. Examples of chromatographic media include environment, derivationally phenyl, butilkoi or octile groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like, Suitable solid carriers include glass beads that tar-based oxide cu is mnia, cellulose resins, agarose pellets, crosslinked agarose granules, polystyrene granules, crosslinked polyacrylamide resin, etc. which are insoluble under the conditions in which they should be used. These carriers can be modified reactive groups, allowing connection of proteins through amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate parts of molecules. Examples of chemical methods of binding include the activation of cyanogenmod, activation, N-hydroxysuccinimide, the activation of the epoxide, the activation of sulfhydryl groups, activation of the hydrazide and carboxyl and amino derivatives for ways of combination using carbodiimide. These and other solid environment are well known and widely used in this field and are available from commercial sources. How binding receptor polypeptides with environments-carriers well known in this field. The choice of a particular method is routine and is determined partly by the properties of the selected media. See, for example, Affinity Chromatographv: Principles & Methods. Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The polypeptides of this invention can be selected through the use of their physical or biochemical properties. For example, adsorption chromatography with immobilized the second metal ion (IMAC) can be used to purify histidine-rich proteins or proteins, having polyhistidine label. Briefly, the gel is first loaded ions of divalent metal with the formation of chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Rich in histidine proteins will be adsorbed on the matrix with different affiniscape depending on the metal ion and will buyouts competitive elution, by lowering the pH or the use of strong chelating agents. Other methods of purification include purification of glycosylated proteins by affinity chromatography with immobilized in pectin and ion exchange chromatography (Methods in Enzvmol., Vol. 182:529-39, "Guide to Protein Purification", M. Deutscher, (ed.), (Acad. Press, San Diego, 1990, pp.) and the use of soluble zalpha11 receptor. In additional embodiments of the present invention can be constructed hybrid interest of the polypeptide and the affinity tag (e.g., malesurvivor protein, immunoglobulin domain) for easy cleaning.

In addition, using the methods described in this region, fused polypeptides or hybrid proteins zalpha11 ligand design using regions or domains zalpha11 ligand of the present invention in combination with areas or domains of other proteins of the family of cytokines person (such as interleukins or GM-CSF), or heterologous proteins (Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur. Opin. Biology, 5:511-5, 1994 and the references). These with the ladies allow you to determine the biological importance of larger domains or areas of interest of the polypeptide. Such hybrids can change the kinetics of the reaction, binding to narrow or expand the substrate specificity or modify tissue and cellular localization of the polypeptide and can be applied to polypeptides of unknown structure.

Fused proteins can be obtained by methods known to experts in this field, by obtaining each component of the fused protein and chemical conjugation. Alternatively, polynucleotide encoding one or more components of the fused protein in the correct reading frame, can be obtained using known methods and expressed using the methods described here. For example, a portion of the spiral or the entire spiral, which imparts a biological function, may be subject to exchange between zalpha11-ligand of the present invention, and functionally equivalent spirals from another family member, such as IL-15, IL-2, IL-4 or GM-CSF. Such components include, but are not limited to, the secretory signal sequence, helix a, b, C and D; loops A/b, b/C, C/D, representing chetyrehspalnyh bundle cytokines. One would expect that such hybrid proteins have biological features that are the same with the polypeptides of this invention or similar characteristics of the polypeptides of this invention or others is now known representing chetyrehspalnyh beam protein family of cytokines families depending on designed fused protein. In addition, such fused proteins can exhibit other properties, as described here.

Standard methods of molecular biology and cloning can be used to exchange the equivalent domains between the zalpha11 polypeptide-ligand and a polypeptide with which they merge. Typically, a DNA segment that encodes a domain of interest, such as described herein helix A-D zalpha11 ligand or another domain, functionally (resectable) linked in reading frame with at least one other segment of DNA encoding an additional polypeptide (for example, a domain or region from another cytokine, such as IL-2 or the like), and embed in suitable expressing vector, as described herein. Usually design DNA construct such that multiple segments of DNA encoding the corresponding regions of the polypeptide that is functionally linked in reading frame to produce a single structure that encodes the entire protein or its functional part. For example, the design of the DNA code from M-end-to-end protein containing a signal polypeptide followed by a Mature protein containing chetyrehspalnyh bundle cytokine that contains the coil And then the coil, then the coil, then the coil D. Such fused proteins can be expressed, isolated and tested for activity is ü, as described here.

The zalpha11 polypeptide-ligand or fragments thereof can also be obtained by chemical synthesis. The zalpha11 polypeptide-ligand can be monomers or multiparae; glycosylated or deglycosylation; paglierani or naegeliana and may contain or may not contain the initiating amino acid residue methionine. For example, polypeptides can be obtained by solid-phase peptide synthesis, for example,described by Merrifield, J. Am. Chem. Soc. 85:2149, 1963.

The activity of molecules of the present invention can be measured using different methods, which measure the proliferation and/or binding to cells expressing zalpha11 receptor. Of particular interest are changes zalpha11 ligand-dependent cells. Suitable cell lines that can be constructed, include IL-3-dependent cell line BaF3 (Pakacios and Steinmetz, Cell 41:727-734, 1985; Mathey-Prevot et al, Mol. Cell. Biol. 6:4133-4135, 1986), FDC-P1 (Hapel et al., Blood 64:786-790, 1984) and Moi (Kiss et al., Leukemia 7:235-240, 1993). Dependent growth factor cell lines can be installed in accordance with published methods (e.g., Greenberger et al., Leukemia Res. 8:363-375, 1984; Dexter et al., in Baum et al., Eds., Experimental Hematology Today. 8thAnn. Mtg. Int. Soc. Esp. Hematol. 1979, 145-156, 1980).

Proteins of the present invention is applicable to stimulate proliferation, activation, differentiation and/or the Indus is tion or inhibit specialized cell functions, cell, involved in the homeostasis of hematopoiesis and immune function. In particular, the polypeptides zalpha11 ligand used for stimulating the proliferation, activation, differentiation, induction or inhibition of specific cell functions of hematopoietic cells lines, including, but not limited to, T cells, b cells, MK-cells, dendritic cells, monocytes and macrophages, and epithelial cells. Proliferation and/or differentiation gemopoeticheskoi cells can be measured in vitro using cultured cells or in vivo introduction of the molecules of the present invention in a suitable animal model. Assays measuring proliferation or differentiation of cells are well known in this field. For example, assays measuring proliferation include such tests as sensitivity to chemical stimuli, for example, to dye neutral red (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990, incl. here as a reference), the incorporation of radioactively labeled nucleotides (Cook et al., Analytical Biochem. 179:1-7, 1989, incl. here as a reference), the inclusion of 5-bromo-2'-dose irradiation on neurogenesis (BrdU) into DNA proliferous cells (Porstmann et al., J. Immunol. Methods 82:169-179, 1985, incl. here as a reference) and the use of salts of tetrazole (Mosmann, J. Immunol. Methods 65:55 to 63. 1983; Alley et al., Cancer Res. 48:589-601. 1988; Marshall et al., Growth Reg. 5:69-84, 1995; Scudiero et al., Cancer Res. 48:4827-4833, 1988; all included here as a researcher is as a reference). The dimensional analysis of differentiation include, for example, measurement of cell surface markers associated with statespecific the expression of tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB. 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses. 161-171, 1989; all incorporated herein by reference).

Molecules of this invention can be analyzed in vivo using viral delivery systems. Examples of viruses for this purpose include adenovirus, herpes virus, retrovirus, vaccinia virus and adeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus, is currently the most investigated vector gene transfer for delivery of heterologous nucleic acids (for review, see T.S. Becker et al., Meth. Cell Biol. 43:161-89, 1994 and J.T. Douglas and D.T. Curiel, Science & Medicine 4:44-53, 1997).

The activity of zalpha11 polypeptide-ligand as a ligand can be measured by the biosensor microphysiometer based on silicon, which measures the rate of extracellular acidification or excretion of protons associated with receptor binding and subsequent physiologic cellular responses. An example device is microphysiometer Cytosensor™formulated Molecular Devices, Sunnyvale, CA. Various cellular responses, such as proliferate cells, ion transport, production of energy, inflammatory response, regulatory and receptor activation, etc. can be measured this way. See, for example, McConnel, N.M. et al., Science 257:from 1906-1912, 1992; Pitchford, S. et al., Meth. Enzvmol. 228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59, 1998; Van Uefde, I. et al., Eur. J. Pharmacol. 346:87-95, 1998.

In addition, zalpha11 ligand can be used to identify cells, tissues, or cell lines which respond to stimulated zalpha11-ligand pathway. Microphysiometer described above, can be used to quickly identify sensitive to the ligand of cells, such as cells that are sensitive to zalpha11-ligand of the present invention. Cells may be cultured in the presence or in the absence of the zalpha11 polypeptide-ligand. Cells that cause a measurable change in extracellular acidification in the presence of zalpha11 ligand, are sensitive to zalpha11-ligand. Such cells or cell lines can be used to identify antagonists or agonists of the zalpha11 polypeptide-ligand, as described above.

In view of the tissue distribution observed for the zalpha11 receptor, agonists (including the natural zalpha11 ligand/substrate/cofactor etc) and/or antagonists have enormous potential in applications such as in vitro and in vivo. Compounds identified as agonists zalpha11 ligand applicable to reproduced the Oia, proliferation, activation, differentiation and/or induction or inhibition of specific cell functions of cells involved in the homeostasis of hematopoiesis and immune function. For example, zalpha11 ligand and compound-agonists applicable as components of specific environments for cell culture and can be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture. Thus, agonists applicable in specific stimulation of growth and/or development of T cells, b cells, NK cells, cytotoxic lymphocytes and other cells of lymphoid and myeloid lines of differentiation in culture.

Antagonists also applicable as research reagents for characterizing sites of interaction of the ligand-receptor. Antagonists applicable for inhibition of reproduction, proliferation, activation and/or differentiation of cells involved in the regulation of haematopoiesis. Inhibitors of the activity of zalpha11 ligand (antagonist zalpha11 ligand) include antibodies against zalpha11 ligand and soluble zalpha11 receptor-ligand, as well as other peptidic and ones agents (including ribozymes).

Zalpha11 ligand can also be used to identify inhibitors (antagonists) of its activity. Test compounds are added to tests described here DL is the identification of compounds inhibiting the activity of zalpha11 ligand. In addition to these described here test samples can be tested for inhibition of the activity of zalpha11 ligand in numerous assays designed to measure receptor binding, stimulation/inhibition of zalpha11 ligand-dependent cellular responses or proliferation expressing zlh11-receptor cells.

The zalpha11 polypeptide-ligand can be expressed in the form of a fused protein with a constant region of the heavy chain of immunoglobulin, usually Fcthe fragment, which contains two constant domain region and does not contain the variable regions. Methods for such mergers are described in U.S. patent No. 5155027 and 5567584. Such mergers usually are secreted as multimeric molecules, where Fc-parts are connected to each other by a disulfide bond, and two non-lg polypeptide are located in close proximity to each other. Mergers of this type can be used, for example, dimerization, increase the stability and half-life existence in vivo, for affinity purification of the ligand, as a tool of analysis in vitro or antagonist. For use in the analysis of these chimeras is associated with the media over Fcdistrict and used in the ELISA format.

Zalpha11 ligand-binding polypeptide can also be used for purification of ligand. This polypeptide immobilized t is enom media such as pellets, of agarose, cross-linked agarose, glass, cellulosic resins, resins based on silicon dioxide, polystyrene, cross-linked polyacrylamide, or like materials that are stable under conditions of use. Methods of binding polypeptides with solid carriers are known in the field and include amine chemistry, activation cyanogenmod, activation, N-hydroxysuccinimide, the activation of the epoxide, sulfhydryl activation and hydrazide activation. The resulting environment is usually prepared in the form of a column and the liquid containing the ligand, is passed through this column once or several times to allow the ligand to contact the receptor polypeptide. Then this ligand elute using changes in salt concentration, chaotropic agents (guanidine-HCl) or pH to disrupt binding of the ligand-receptor.

The test system, which uses legendbase.ui receptor (or an antibody, one member of the pair complement/anticomplement) or its binding fragment, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) can be advantageously used. Such a receptor, antibody, member of the pair complement/anticomplement or fragment immobilized on the surface of a receptor chip. The application of this device is described Karlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. This cocktail recipes. is, antibody member of a couple or a fragment covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within a flow cell. The test sample is passed through the cell. If the ligand epitope or the opposite member of the pair complement/anticomplement present in this sample, it will bind to the immobilized receptor, an antibody or a member of a pair, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows you to define speed on and off (on - and off-rate), which can be calculated the binding affinity of and the estimation of the stoichiometry of binding. Alternatively, binding of the ligand-receptor can be analyzed using SELDI(TM) (Ciphergen, Inc., Palo Alto, CA).

Landscapemode receptor polypeptides can also be used in other systems known in the field. Such systems include analysis of Scatchard to determine the affinity of binding (see Scatchard, Ann. NY Acad. Sci. 51:660-72, 1949) and calorimetric analyses (Cunningham et al., Science 253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

The zalpha11 polypeptide-ligand can also be used to generate antibodies that bind to the epitopes, PE is the Chida or polypeptides zalpha11 ligand. The zalpha11 polypeptide-ligand or fragment serves as an antigen (immunogen) to inoculation of the animal and the induction of immune responses. Specialist with expertise in this area will be clear that the antigens, epitope-bearing polypeptides contain a sequence of at least 6, preferably at least 9, and more preferably at least about 15 to 30 contiguous amino acid residues of the polypeptide zalpha11 ligand (e.g., SEQ ID NO:2). Included polypeptides containing a large part of the zalpha11 polypeptide-ligand, i.e. from 30 to 100 residues to the full length of this amino acid sequence. Antigens or immunogenic epitopes can also include an attached label, adjuvants and carriers described herein. Suitable antigens include the zalpha11 polypeptide-ligand encoded by SEQ ID NO:2 from the number of amino acids 32 to amino acids 162 or a fragment of contiguous amino acids 9-131. Other suitable antigens include full-sized and Mature zalpha11 ligand, and helices A-D and individual or multiple helix a, b, C and D patterns chetyrekhmernogo beam zalpha11 ligand described herein. Preferred peptides to use as antigens are hydrophilic peptides such as foretold by the person skilled in the art from the graph hydrophobicity as described ZV is camping, for example, amino acid residues 114-119, 101-105, 126-131, 113-118 and 158-162 SEQ ID NO:2.

Antibodies of the immune response generated by inoculation of an animal with these antigens can be isolated and purified as described above. Methods of acquisition and allocation of polyclonal and monoclonal antibodies are well known in the art (see, for example, Current Protocols in Immunology. Cooligan, et al., (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual. Second Edition. Cold Spring Harbor, NY, 1989 and Hurrell, J.G.R., Ed., Monoclonal Hvbridoma Antibodies: Techniques and Applications. CRC Press, Inc., Boca Raton, FL, 1982).

As should be obvious to a person of ordinary skill in this field, polyclonal antibodies can be obtained by inoculation of various warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice and rats, the zalpha11 polypeptide-ligand or its fragment. The immunogenicity of the polypeptide zalpha11 ligand can be increased by applying an adjuvant, such as alum (aluminum hydroxide) or a full or partial beta-blockers. Polypeptides that are applicable for immunization also include fused (hybrid) polypeptides, such as hybrids zalpha11 ligand or part of the immunoglobulin polypeptide or with malesurvivor protein. Polypeptide immunogen can be a full-length molecule or a part of it. If this part of the polypeptide is "a hapten-p is such", this part can preferably be connected or linked to a macromolecular carrier (such as hemocyanin fissurella (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

In the application here, the term "antibodies" includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigennegative fragments, such as proteolytic F(ab')2- and Fab-fragments. Also included genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single-chain antibodies and the like, and synthetic antigennegative peptides and polypeptides. Antibodies of the person can be "humanized" (humanitarian) by grafting the CDR is not the person on the framework and constant region of human immunoglobulin or the inclusion of a complete variable domains of a person (not necessarily a "wrapping" them with a human-like surface by replacement of exposed residues, resulting in a "tiled" antibody). In some cases, "humanized" antibodies can save the remnants of immunoglobulin could frame the domain of the variable region of a person to enhance the characteristics of the correct binding. Through humanitarian" antibodies can be increased biological the second half-period of existence in the body, and the potential for adverse immune response when administered to humans is reduced. In addition, human antibodies can be produced in transgenic animals, non-human, which were designed to contain genes of human immunoglobulin, as described in WIPO Publication WO 98/24893. Preferably, the endogenous immunoglobulin genes in these animals were inactivated or eliminated, for example, homologous recombination.

Antibodies are considered as specifically binding if: 1) they exhibit a threshold level of binding activity, and 2) they have no significant cross-reactivity with related polypeptide molecules. The threshold level of binding is determined if antibodies against zalpha11 ligand described herein are contacted with the polypeptide, peptide or epitope zalpha11 ligand with an affinity at least 10-fold greater than the binding affinity of control (not zalpha11-ligand) polypeptide. Preferably, these antibodies showed binding affinity of (Kand) 106M-1or greater, preferably 107M-1or greater, more preferably 108M-1or greater, and most preferably 109M-1or greater. The binding affinity of antibodies can be readily determined by a specialist with Oba is Noah qualifications in this area, for example, using analysis of Scatchard (Scatchard. Ann. NYAcad. Sci. 51:660-672, 1949).

The fact that antibodies against zalpha11 ligand have no significant cross-reactivity with related polypeptides shown, for example, the detection of this antibody polypeptide zalpha11 ligand, but not known related polypeptides using a standard Western blot analysis (Ausubel et al., ibid.). Examples of known related polypeptides include orthologues and paralogy and similar well-known members of this family of proteins. Screening can also be done using zalpha11 ligand is not human and mutant polypeptides zalpha11 ligand. In addition, antibodies can be subjected to "screened against" known related polypeptides for the selection of a population that specifically binds to a polypeptide zalpha11 ligand. For example, antibodies, induced to zalpha11-ligand, adsorb related polypeptides attached to insoluble matrix; antibodies specific to zalpha11 ligand will flow through the matrix under the proper buffer conditions. Screening allows you to isolate polyclonal and monoclonal antibodies that do not have cross-reactivity with known related polypeptides (Antibodies: A Laboratory Manual. Hariow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology. Cooligan, et al. (eds.), National Institutes o Health, John Wiley and Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in this area (see Fundamental Immunology. Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43:1-98, 1988; Monoclonal Antibodies: Principles and Practice. Coding, J.W. (eds.), Academic Press Ltd.. 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984). Specific binding of antibodies against zalpha11 ligand can be defined in many ways known in this area and are described below.

A variety of assays known to those with ordinary skill in this field can be used to detect antibodies that specifically bind proteins and polypeptides zalpha11 ligand. Examples of the analyses described in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Typical examples of such analyses include: competitive immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot analysis, inhibitory or competitive analysis and composite analysis. In addition, antibodies can be subjected to screening for binding to the protein or polypeptide of the wild type compared to the mutant protein or polypeptide zalpha11 ligand.

Antibodies to zalpha11 can be used for tagging cells expressing zalpha11 ligand; to highlight zalpha11 ligand affinity purification; for diagnostic tests to determine the levels in the bloodstream p is dipeptides zalpha11 ligand; for the detection or quantitative determination of soluble zalpha11 ligand as a token hidden pathology or disease; in analytical methods using FACS; for screening expression libraries; for generating antiidiotypic antibodies and as neutralizing antibodies or as antagonists to block the activity of zalpha11 ligand in vitro and in vivo. Suitable direct tags (markers) or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags (markers) or labels can withdraw in the first place the use of pairs of Biotin-avidin or other pairs complement/anticomplement as intermediate products. These antibodies may be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates can be used for diagnostic or therapeutic applications in vivo. In addition, antibodies to zalpha11-ligand or its fragments can be used in vitro to detect denatured zalpha11 ligand or its fragments in the analysis, for example, Western-blots or other assays known in this field.

Suitable detected molecules may be directly or indirectly attached the to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like are Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (e.g., diphtheria toxin, saporin, Pseudomonas exotoxin, ricin, abrin etc), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through, for example, chelating portion of the molecule). The polypeptides or antibodies can also be conjugated with cytotoxic drugs such as adriamycin. For indirectly attaching a detectable or cytotoxic molecules detected this or cytotoxic molecule can be conjugated with a member couples the complement/anticomplement, where another member is associated with the polypeptide or part of the antibody. For these purposes, an example of complementary/anticomplementary pair is Biotin/streptavidin.

The binding polypeptides can also act as "antagonists" zalpha11 ligand to block the binding of zalpha11 ligand and signal transduction in vitro and in vivo. These polypeptides acting against binding zalpha11 ligand, were p is iminime for inhibiting the activity of zalpha11 ligand or binding protein.

Hybrid proteins with polypeptide-toxin or hybrid proteins with antibody-toxin can be used for targeted inhibition or destruction of cells or tissue (e.g., for treatment of cancer cells or tissues). Alternatively, if the polypeptide has multiple functional domains (i.e. activating domain or receptornegative domain, plus the target domain), a hybrid protein consisting of the target domain may be suitable for the direction of the detectable molecule, cytotoxic molecule or complementary molecules of interest in the cell type or tissue. In cases where a hybrid protein with a single domain includes complementary molecule, anticomplementary molecule can be conjugated with a detectable or cytotoxic molecule. Thus, these hybrid proteins containing the domain-complementary molecule, are characteristic of a particular cell type targeting media to kletka/tissue-specific delivery typical for this type of cell conjugates anticomplementary-detected/cytotoxic molecules.

Hybrid proteins zalpha11 ligand is a cytokine or hybrid proteins with antibody-cytokine can be used for enhancing in vivo killing of target tissues (for example, cancers of the blood and bone MH is (a), if the zalpha11 polypeptide-ligand or antibody against zalpha11 ligand affects hyperproliferative blood cell or bone marrow (see, generally, Hornick et al., Blood 89:4437-47, 1997). Described fused proteins allow you to target the cytokine to a desired site of action, by providing this higher local concentration of cytokine. Suitable polypeptides zalpha11 ligand or antibodies against zalpha11 ligand amaze undesirable cell or tissue (i.e., the tumor or a leukemia), and the fused cytokine Mediaset improved lysis of target cells effector cells. Suitable cytokines for this purpose include, for example, interleukin 2 and granulocyte-macrophage colony-stimulating factor (GM-CSF).

Differentiation is a progressive and dynamic process that begins pluripotent stem cells and ending finally differentiated cells. Pluripotent stem cells that can be regenerated without commitirovannah to the line of differentiation, Express a set of differentiation markers that are lost when you commitirovannah in relation to the sequence of cell generations. Precursor cells Express a set of markers of differentiation, which can continue to be expressed or might not continue to be expressed by measures of the progression of these cells towards the direction of differentiation to achieve maturity. Markers of differentiation, which is expressed exclusively in Mature cells are usually functional properties, such as cellular products, enzymes for the production of cell products and receptors. The stage of differentiation of the cell population is subjected to monitoring by identifying markers that are present in this cell population.

There is evidence to suggest that factors that stimulate specific types of cells in the direction towards the ultimate differentiation or dedifferentiate affect the whole cell population is derived from a common precursor or a common stem cell. Thus, this invention includes the stimulation or inhibition of proliferation of lymphoid cells, hematopoietic cells and epithelial cells.

Zalpha11 ligand was isolated from tissue known that she has an important immunological function and which contains cells that play a role in the immune system. Zalpha11 ligand is expressed in CD3+ selected activated peripheral blood cells, and it has been shown that the expression of zalpha11 ligand is increased after activation of T cells. In addition, the results of the experiments described in the examples section, demonstrate that the polypeptides of the present invention have an effect on the growth/RA is mnozenie and/or differentiated state of NK-cells or precursors of NK-cells. Additional evidence demonstrates that zalpha11 ligand affects the proliferation and/or differentiation of T-cells and b-cells in vivo. Factors which stimulate the proliferation gemopoeticheskoi precursor cells and activate Mature cells, in General, known. NK-cells are sensitive only to IL-2, but the proliferation and activation usually requires additional growth factors. For example, it was shown that IL-7 and Steel factor (c-kit-ligand) was required for the formation of colonies of NK-cell precursors. IL-15+IL-2 in combination with IL-7 and Steel factor was more effective (Mrozek et al., Blood 87:2632-2640, 1996). However, unidentified cytokines may be necessary for the proliferation of specific subpopulations of NK-cells and/or NK-cell precursors (Robertson et al., Blood 76:2451-2438, 1990). Composition containing zalpha11 ligand and IL-15 stimulates NK precursor cells and NK-cells, indicating that this composition is more potent than previously described factors and combinations of factors.

Tests to measure differentiation include, for example, measurement of cellular markers associated with statespecific the expression of tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses. 161-171, 1989; vkluchennosti as a reference). Alternatively, the zalpha11 polypeptide-ligand itself may serve as an additional marker of cell surface or secreted marker associated with stagespecific expression of tissue. By itself, direct measurement of the zalpha11 polypeptide-ligand or loss of its expression in the tissue as differentiation may serve as a marker for differentiation of tissues.

Similarly, direct measurement of the zalpha11 polypeptide-ligand or loss of its expression in tissues can be performed in a tissue or in cells when they are exposed to the progression of the tumor. Increase the invasiveness and motility of cells or the increase or loss of expression of zalpha11 ligand in precancerous or cancerous condition in comparison with normal tissue can serve as diagnostic tools for transformation, invasion and metastasis in tumor progression. The mere knowledge of the state of progression of the tumor or metastasis will help the physician to choose the most appropriate therapy and aggressiveness of treatment for individual cancer patient. Ways to measure the increase or loss of expression (either mRNA or protein) are well known in this field and is described here and can be used for the expression of zalpha11 ligand. For example, the appearance or disappearance of polypeptides, adjustable lighting angle is related cell motility, can be used to aid in diagnosis and prognosis of prostate cancer (Banyard, J. and Zetter, B.R., Cancer and Metast. Rev. 17:449-458, 1999). As an effector of cell motility, increase or loss of expression of zalpha11 ligand can serve as a diagnostic tool for non-Hodgkin's lymphoma, b-cell, epithelial, geopositions and other cancers.

In addition, the activity and effect of zalpha11 ligand on the development and metastasis of the tumor can be measured in vivo. Several models of syngeneic mice have been developed to study the effect of polypeptides, compounds or other treatments on tumor development. In these models, tumor cells, passiruete in culture, implanted in mice of the same strain as the donor tumors. These cells will develop into tumors that have similar characteristics in the recipient mice, and metastasis will also take place in some of these models. Suitable tumor models for the research of the authors include the Lewis lung carcinoma (ATSS No. CRL-1642) and B16 melanoma (ATSS No. CRL-6323) among others. Both are commonly used tumor lines, syngeneic relatively mice C57BL6/J, which are easily amenable to cultivation and manipulation in vitro. Tumors resulting from implantation of any of these cell lines capable IU stateroute in the lung in mice C57BL6/J. The model of Lewis lung carcinoma has recently been used in mice to identify an inhibitor of angiogenesis (O'reilly MS, et al., Cell 79:315-328, 1994). Mice C57BL6/J is treated with an experimental agent or by injection once a day recombinant protein, agonist or antagonist, or a single injection of recombinant adenovirus. Three days after this treatment, 105-106cells are implanted under the skin of the back. Alternatively, these cells can be infected with recombinant adenovirus, such as expressing zalpha11 ligand, before implantation so that the protein is synthesized at the tumor site or intracellularly, and not systematically. Mice usually develop visible tumors within 5 days. Tumors give rise in the period up to 3 weeks, and during this period they can reach the size of 1500-1800 mm3in the control treated group. Tumor size and body weight are careful monitoring during this experiment. While killing the tumor was removed and weighed together with the liver and lungs. It was shown that the weight of the lung correlates well with metastatic tumor load. As an additional measure considered metastases to the lung surface. Then the tumor, lungs and liver are prepared for histopathological examination, immunohistochemistry and in situ guy is realizatsii using, known in this field and are described here. Thus can be estimated influence of this expressed polypeptide, for example, zalpha11 ligand, on the ability of the tumor to recruit vasculature and undergo metastasis. In addition, along with the use of adenovirus, the implanted cells can be temporarily transliterowany zalpha11-ligand. The use of stable zalphall-ligand-transfectants, and the use of inducible promoters to activate the expression of zalpha11 ligand in vivo is known in this field and can be used in this system to assess the induction of metastasis zalpha11-ligand. In addition, purified zalpha11 ligand or air-conditioned zalphall-ligand environment can be directly injected in to this mouse model and, therefore, used in this system. In respect of General references, see O'reilly MS, et al., Cell 79:315-328, 1994 and Rusciano d, et al., Murine Models of Liver Metastasis. Invasion Metastasis 14:349-361, 1995.

Zalpha11 ligand will be applicable in the treatment of carcinogenesis and, therefore, could be used in the treatment of cancer. Zalpha11 ligand inhibits stimulated IL-4 proliferation stimulated by anti-lgM normal b-cells, and a similar effect is observed in the lines of b-cell tumors, which suggests that there may be a therapeutic benefit in the treatment of patients zalpha11-ligand induction cleto the b-cell tumors in less proliferative state. This ligand could be implemented in combination with other existing agents, including with conventional chemotherapeutic agents, and immunomodulators such as interferon alpha. It was shown that alpha/beta interferons are effective in the treatment of some leukemias and models of animal diseases and inhibiting the growth effects of interferon-alpha and zalpha11 ligand and are additive in relation to the at least one derived from a b-cell tumor cell line.

This invention provides a method of reducing proliferation of a neoplastic (tumor) b - and T-cells, involving the administration to a mammal with a B - or T-cell neoplasms amount of the composition zalpha11 ligand sufficient to reduce proliferation of neoplastic B - or T-cells. In other embodiments, the composition may contain at least one other cytokine selected from the group consisting of IL-2, IL-15, IL-4, GM-CSF, Flt3-ligand or stem cell factor.

In another aspect, the invention provides a method of reducing proliferation of neoplastic B - or T-cells, involving the administration to a mammal with a B - or T-cell neoplasms number antagonist zalpha11 ligand sufficient to reduce proliferation of neoplastic B - or T-cells. In other embodiments, the composition may win at least one other cytokine, selected from the group consisting of IL-2, IL-15, IL-4, GM-CSF, Flt3-ligand or stem cell factor. In addition, the antagonist zalpha11 ligand may be fused protein ligand/toxin.

Merged toxin zalpha11 ligand-saporin can be used against a similar series of leukemias and lymphomas, expanding the range of leukemias that can be treated zalpha11-ligand. Mediated merged toxin activation zalpha11 receptor provides two independent means for inhibiting the growth of target cells, and the first tool is identical to the effects observed with the use of one ligand, and the second is due to the delivery of the toxin through the internalization of the receptor. Limited lymphoedema pattern of expression of zalpha11 receptor suggests that conjugate the ligand-saporin can be tolerated by patients.

When the treatment of malignant zabolevanii includes bone marrow transplantation or stem cells, zalpha11 ligand may be useful in the amplification reaction, graft versus tumor. Zalpha11 ligand stimulates the generation of lytic MK-cells from precursors in the bone marrow and stimulates the proliferation of T cells after activation of antigen receptors. Thus, when patients receive allogeneic bone marrow transplants, zalpha11 ligand will enhance the generation of antitumor responses, with details what Zia or without infusion of donor lymphocytes.

Tissue distribution of the receptor-specific cytokine gives a strong indication of the potential sites of action of this cytokine. Northern analysis zalpha11 receptor revealed transcripts in the spleen, thymus, lymph node, bone marrow and peripheral blood leukocytes of man. Specific types of cells were identified as cells expressing zalpha11 receptor, and strong signals were observed in the reaction of the mixed culture of lymphocytes (SCR) and in cells Raji lymphoma Bernita. Two lines of monocytes, TPR-1 (Tsuchiya et al., Int. J. Cancer 26:171-176. 1980) and U937 (Sundstrom et al., Int. J. Cancer 17:565-577, 1976) were negative.

Zalpha11 receptor is expressed at relatively high levels in the SCR, in which mixed mononuclear cells of peripheral blood (PBMNC) from two individuals, which leads to mutual activation. Detection of high levels of transcript in this SCR, but not in resting T - or b-cell populations, suggests that the expression of zalpha11 receptor can be induced in one or more cell types during activation. Activation of isolated populations of T - and b-cells can be artificially achieved by stimulation of the cells with the help of PMA and ionomycin. When the exposure past the sorting of cells with these conditions the activation levels of the transcript zalpha11 receptor was increased in both cell types, confirming the role of DL is that zalpha11 receptor and ligand in immune reactions, in particular in the autocrine and paracrine outbreaks of T-cells and b-cells during activation. Zalpha11 ligand may also play a role in the reproduction of more primitive predecessors involved in lymphopoiesis.

It was found that zalpha11 receptor was present at low levels in resting T and b cells and is positively regulated during activation in both cell types. Interestingly, b cells also negatively regulated by this mediator faster than T-cells, suggesting that the signal amplitude and timing of the blanking signal are important for appropriate regulation In cells.

In addition, a large proportion of the cells own layer of the intestinal mucosa (lamina propria) finds a positive hybridization signals with the zalpha11 receptor. This fabric consists of a mixed population of lymphoid cells, including activated CD4+ T cells and activated b cells. Immune dysfunction, including chronic activation of the immune reaction of the mucous membrane, plays a role in the etiology of Crohn's disease; abnormal response to proinflammatory cytokines and/or production of proinflammatory cytokines is also estimated factor (Braegger et al., Annals Allergy 72:135-141, 1994; Sartor RB Am. J. Gastroenterol. 92:53-11S, 1997). Zalpha11 ligand together with IL-15 breeds of NK cells from cells predshestvuyuschego brain and enhances the effector function of NK cells. Zalpha11 ligand will also costimulate Mature b cells stimulated with antibodies to CD40, but inhibits proliferation of b-cells to signals transmitted through the IgM. Zalpha11 ligand enhances the proliferation of T-cells together with a signal through the receptor of T cells, and overexpression in transgenic mice leads to lymphopenia and proliferation of monocytes and granulocytes. These pleiotropic effects zalpha11 ligand suggest that it may provide therapeutic primenimosti for a wide range of diseases arising from defects in the immune system, including (but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), heavy pseudoparallelism gravis and diabetes. It is important to note that these diseases are the result of a complex network of immune dysfunction (e.g., SLE is a manifestation of defects in T-and B-cells) and immune cells depends on the interaction with each other for the induction of a strong immune response. Thus, zalpha11 ligand (or antagonist of the ligand), which can be used to manipulate more than one type of immune cells, is an attractive therapeutic candidate for intervention in numerous stages of the disease.

The polypeptides and proteins of this invention can be used is carried out ex vivo, for example, autologous culture of bone marrow. Briefly, bone marrow is removed from the patient before chemotherapy or organ transplantation and treated with zalpha11-ligand, optionally in combination with one or more other cytokines. The treated bone marrow is then returned to the patient after chemotherapy to speed up the recovery of bone marrow or after transplantation for the suppression reaction, graft versus host. In addition, proteins of this invention can also be used for propagation ex vivo bone marrow or precursor cells of peripheral blood (RVRS). Before treatment, the bone marrow can be stimulated with stem cell factor (SCF) for release early precursor cells in the blood flow of peripheral blood. These precursor cells can be collected and concentrated from peripheral blood and then treated in culture zalpha11-ligand, optionally in combination with one or more other cytokines, including, but not limited to, SCF, IL-2, IL-4, IL-7 or IL-15 for differentiation and proliferation in lymphoid culture of high density, which are then returned to the patient after chemotherapy or transplantation.

This invention provides a method of propagation of hematopoietic cells and hematopoietic precursors cells, OEM home button Flex cable is trevose cultivation of bone marrow cells or peripheral blood cells with a composition, contains the number of zalpha11 ligand sufficient to obtain the increase in the number of lymphoid cells in the bone marrow cells or peripheral blood cells as compared with bone marrow cells or peripheral blood, cultured in the absence of zalpha11 ligand. In other embodiments, the hematopoietic cells and hematopoietic cell precursors are lymphoid cells. In another embodiment, the lymphoid cells are MK-cells or cytotoxic T cells. In addition, the composition may also contain at least one other cytokine selected from the group consisting of IL-2, IL-15, IL-4, GM-CSF, Flt3 and stem cell factor.

Alternative zalpha11 ligand can activate the immune system, which would be important in boosting immunity to infectious diseases, the treatment of patients with impaired immunity, such as HIV+ patients, or in improving vaccines. In particular, stimulation of the zalpha11-ligand or reproduction of NK-cells or their precursors could provide therapeutic benefit in the treatment of viral infections and as an antitumor factor. It is believed that NK cells play a major role in the elimination of metastatic tumor cells, and patients with metastatic disease, and solid tumors have reduced levels of activity of NK-cells (Whiteside et al., Curr. Top. Environ. Immunol. 230:221-224, 1998). Beneath is mainly stimulation of the zalpha11-ligand immune response against viral and non-viral pathogenic agents (including bacteria, protozoa and fungi) would give a therapeutic benefit in the treatment of such infections by inhibiting the growth of infectious agents. Determining, directly or indirectly, the levels of the pathogen or antigen, such as a tumor cell present in the body, can be achieved in a number of ways known in this field and are described here.

The invention includes a method of stimulating an immune response in a mammal exposed to an antigen or pathogen, providing stages: (1) determining directly or indirectly the level of antigen or pathogen present in the specified mammal; (2) introducing a composition comprising the polypeptide zalpha11 ligand in a pharmaceutically acceptable carrier; (3) determining directly or indirectly the level of antigen or pathogen present in the specified mammal; and (4) comparing the level of antigen or pathogen in stage 1 with the level of antigen or pathogen in stage 3, and the change in this level is a sign of stimulation the immune response. In another embodiment, the composition of the zalpha11 ligand is administered repeatedly. In other embodiments, the antigen is a b-cell tumor; virus; parasite or bacterium.

In another aspect of this is sabreena provides a method of stimulating the immune response in the mammal, exposed to the antigen or pathogen, providing stages: (1) determining the level of antigen - or pathogen-specific antibody; (2) introducing a composition comprising the polypeptide zalpha11 ligand in a pharmaceutically acceptable carrier; (3) determining the level of antigen - or pathogen-specific antibodies after injection; (4) comparing the level of antibody in stage (1) with the level of antibodies in stage (3), and the increase in antibody level is indicative of stimulating an immune response.

Polynucleotide encoding polypeptides zalpha11 ligand applicable in the methods of gene therapy where it is desired to increase or inhibit the activity of zalpha11 ligand. If a mammal has a mutated or missing gene zalpha11 ligand, this gene zalpha11 ligand can be introduced into cells of the mammal. In one embodiment, the gene encoding the zalpha11 polypeptide-ligand is administered in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), human papilloma virus, the virus of Epstein-Barr (EBV), adenovirus, adeno-associated virus (AAV), etc. are Preferred defective viruses, which are completely or almost completely devoid of viral genes. Defective virus is not infectious after introduction into the cell. The use of defective viral the vectors makes possible the introduction into the cells in a specific, localized area, without fears that this vector can infect other cells. Examples of particular vectors include, but are not limited to, the vector of defective herpes simplex virus 1 (HSV1) (Kaplitt et al., Molec. Cell. Neurosci., 2:320-30, 1991), attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30, 1992), and a defective adeno-associated virus vector (Samulski et al., J. Virol., 61: 3096-101, 1987; Samulski et al., J. Virol., 63:3822-8, 1989).

In another embodiment, the gene zalpha11 ligand can be introduced in a retroviral vector, for example, as described in Anderson et al., U.S. Patent No. 5399346; Mann et al., Cell. 33:153, 1983; Temin et al., U.S. Patent No. 4650764; Temin et al., U.S. Patent No. 4980289; Markowitz et al., J. Virol. 62:1120, 1988; Temin et al., U.S. Patent No. 5124263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al.; and Kuo et al., Blood, 82:845, 1993. Alternatively, the vector can be introduced by lipofectin in vivo using liposomes. Synthetic cationic lipids can be used to produce liposomes for transfection in vivo gene encoding a marker (Felgner et al., Proc. Natl. Acad. Sci. USA. 84, 7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988). Using lipofectin for the introduction of exogenous genes in specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells is one of the area advantages. More specifically, the direction transfer the AI in specific cells represents one of the area advantages. For example, the direction transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the immune system, pancreas, liver, kidney and brain. Lipids may be chemically bonded to other molecules for targeting. Targeted peptides (e.g., hormones or neurotransmitters, proteins, such as antibodies, or ones molecule can be linked to liposomes chemically.

You can remove the target cells from the body; to enter this vector in the form of "naked" DNA plasmids and then re-implanting these transformed cells in the body. Naked DNA vectors for gene therapy can be introduced into the desired cells are the owners of the methods known in this field, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE-dextranomer method, the calcium phosphate precipitation using gene shotgun (shotgun-way) or using a vector-vector DNA. See, for example, Wu et al., J. Biol. Chem. 267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

Antisense methodology can be used to inhibit transcription of the gene zalpha11 ligand, for example, for inhibition of cell proliferation in vivo. Construct polynucleotides that are complementary to a segment encoding zalpha11 ligand of polynucleotide (for example, Pauline is leotide, presented in SEQ ID NO:1) for binding to the coding zalphall-ligand mRNA and inhibit translation of such mRNA. Such antisense polynucleotide applicable for the inhibition of the expression of the encoding polypeptides zalpha11 ligand genes in cell culture or in a subject.

Mouse, "engineered" for gene expression of zalpha11 ligand, called "transgenic mice," and mice that found a complete absence of gene function zalpha11 ligand, called "mice with knockout (gene), can be obtained (Snouwaert et al., Science 257: 1083, 1992; Lowell et al., Nature 366: 740-42, 1993; Capecchi, M.R., Science 244:1288-1292, 1989; Palmiter, R.D. et al., Annu Rev Genet. 20:465-499, 1986). For example, transgenic mice that sverkhekspressiya zalpha11 ligand, either ubiquitously or under the control of tissue-specific promoter or restrukturovaneho against tissue promoter, can be used to determine, determines whether the overexpression of a specific phenotype. For example, overexpression of the zalpha11 polypeptide-ligand wild-type fragment of the polypeptide or mutant may alter normal cellular processes, leading to a phenotype that identifies a tissue in which expression of zalpha11 ligand is functionally relevant and may indicate a therapeutic target for zalpha11 ligand, its agonists or antagonists. For example, a preferred transgenic mouse to "to whom struisbaai" is the mouse which sverkhekspressiya zalpha11 ligand (amino acid residues 32-162 SEQ ID NO:2). In addition, this overexpression may lead to the phenotype, which shows a similarity with human diseases. Similarly, mice with knockout of zalpha11 ligand can be used to determine where zalpha11 ligand is absolutely required in vivo. The phenotype of mice with a knockout predicts effects in vivo, which may have antagonist zalpha11 ligand, such as described here antagonists. cDNA of murine or human zalpha11 can be used to generate mice with knockout of the gene. These mice can be used to study gene zalpha11 ligand and proteins encoded by them, in the system in vivo and can be used as models in vivo to the corresponding human diseases. In addition, expression of transgenic mice antisense polynucleotides zalpha11 ligand or anti-zalpha11 ligand ribozymes described herein, can be used similarly, expression of the transgenic mice described above. Research can also be carried out through the introduction of purified protein zalpha11 ligand.

For pharmaceutical applications, the proteins of the present invention is prepared for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. Biologically active is aliphatic or conjugates of antibodies, described herein may be delivered intravenously, intraarterially or vnutriplitnogo or can be entered locally at the intended site of action. Intravenous administration can be bolus injection or by infusion over a regular period of time of one to several hours. Typically, the pharmaceutical compositions will include protein zalpha11 ligand in combination with a pharmaceutically acceptable carrier, such as saline, buffered saline, 5% dextrose in water or the like, the Composition can optionally contain one or more fillers, preservatives, solubilization, bafarawa agents, albumin to prevent loss of protein on the surfaces of the bottles, etc. Methods for making compositions are well known in this field and are described, for example, in Remington: The Science and Practice of Pharmacy. Gennaro, ed., Mack Publishing Co., Easton, PA, 19thed., 1995. Therapeutic doses will generally be in the range from 0.1 to 100 μg/kg of body weight per day, preferably 0.5 to 20 mcg/kg per day, with the exact dose must be determined by a physician in accordance with accepted standards, taking into account the nature and gravity of the subject to treatment status, patient, etc. dose Determination is within the ordinary skill of a person skilled in the art. Proteins can be entered for urgently needed treatment, is and for one week or less, often over a period of one to three days, or can be entered into a long-term treatment for several months or years. Typically a therapeutically effective amount of a soluble receptor polypeptide zalpha11 ligand is sufficient to obtain a clinically meaningful effect.

Further, the invention is illustrated by the following non-restrictive examples.

EXAMPLES

Example 1

Construction of chimeras MPL-zalpha11 polypeptide: extracellular and TM-domain of the MPL, merged with the intracellular domain signal zalpha11

The extracellular and transmembrane domains of the murine MPL receptor was isolated from a plasmid containing the murine MPL receptor (plasmids PHZ1/MPL) using PCR with primers ZC17212 (SEQ ID NO:5) and ZC19914 (SEQ ID NO:6). Reaction conditions were as follows: 95°C for 1 min; 35 cycles at 95°C for 1 minute, 45°C for 1 minute, 72°C for 2 min; then 72°C for 10 minutes; then soaking in 10°C. the PCR product was subjected to electrophoresis on 1% agarose, low melting point (Boehringer Mannheim, Indianapolis, IN) and a fragment of MPL-receptor approximately 1,5 TPN was isolated using a kit for the extraction of Qiaquick gel™ Gel Extraction Kit (Qiagen) according to the manufacturer's instructions.

The intracellular domains of the human zalpha11 was isolated from a plasmid containing to the NC zalpha11 receptor using PCR with primers ZC19913 (SEQ ID NO:8) and ZC20097 (SEQ ID NO:9). Polynucleotide sequence corresponding to the coding zalpha11 receptor sequence shown in SEQ ID NO:7, and the corresponding amino acid sequence shown in SEQ ID NO:115. Reaction conditions were the same as described above. The PCR product was subjected to electrophoresis on 1% agarose, low melting point (Boehringer Mannheim) and the zalpha11 fragment of approximately 900 TPN was isolated using a kit for the extraction of Qiaquick gel according to the manufacturer's instructions.

Each of the selected fragments described above were mixed at a volume ratio of 1:1 and used in PCR reactions using ZC17212 (SEQ ID NO:5) and ZC20097 (SEQ ID NO:9) to create chimeras MPL-zalpha11. Reaction conditions were as follows; 95°C for 1 min; 35 cycles at 95°C for 1 minute; 55°C for 1 minute, 72°C for 2 min; then 72°C for 10 minutes; then soaking in 10°C. Complete the PCR product was subjected to electrophoresis on 1% agarose, low melting point (Boehringer Mannheim) and chimeric fragment of MPL-zalpha11 approximately 2.4 TPN was isolated using a kit for the extraction of gels Qiaquick (Qiagen) according to the manufacturer's instructions. Chimeric fragment of MPL-zalpha11 were digested EcoRI (BRL) and Xbal (Boehringer Mannheim) according to the manufacturer's instructions. All the cleavage product was subjected to electrophoresis on 1% agarose, low melting point (Boehringer Manheim) and split Chimera MPL-zalpha11 was isolated using a kit for the extraction of gels Qiaquick (Qiagen) according to the manufacturer's instructions. The obtained split Chimera MPL-zalpha11 built in expressing vector, as described below.

Recipient expressing vector pZP-5N was digested EcoRI (BRL) and Hindlll (BRL) according to the manufacturer's instructions, and purified on a gel as described above. This vector fragment was combined with the split EcoRI and Xbal Chimera MPL-zalpha11 allocated, as described above, and Xbal/Hindlll-linker fragment in a ligation reaction. Ligation was performed with T4 ligase (BRL) at 15°With during the night. A sample of the ligation was electroporative in electrocompetent relatively DH10B ElectroMAX™ cells of E. coli (25 μf, 200 Ω, 2.3 In). Transformants were sown on plates with LB+ampicillin and individual colonies were screened using PCR to confirm the Chimera MPL-zalpha11 using ZC17212 (SEQ ID NO:5) and ZC20097 (SEQ ID NO:9) using the PCR conditions described above.

The confirmation sequence chimeras MPL-zalpha11 performed using analysis sequence using the following primers: ZC12700 (SEQ ID NO:10), ZC5020 (SEQ ID NO:11), ZC6675 (SEQ ID NO:12), ZC7727 (SEQ ID NO:13), ZC8290 (SEQ ID NO:14), ZC19572 (SEQ ID NO:15), ZC6622 (SEQ ID NO:16), ZC7736 (SEQ ID NO:17) and ZC9273 (SEQ ID NO:18). Insert (insert) was approximately 2.4 TPN and was full.

Example 2

Proliferation-based chimeras MPL-zalpha11 in F3-test using Alamar Blue

A. Construction of BaF3 cells expressing the Chimera MPL-zalpha11

BaF3, interleukin-3- (IL-3)for isimu preliminay cell line, derived from murine bone marrow (Palacios and Steinmetz, Cell 41:727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6:4133-4135, 1986), was maintained in complete medium (RPMI medium (JRH Bioscience Inc., Lenexa, KS)supplemented with 10% V / V heat inactivated fetal calf serum, 2 ng/ml murine IL-3 (mlL-3) (R & D, Minneapolis, MN), 2 mm L-glutaMax-1™ (Gibco BRL), 1 mm sodium pyruvate (Gibco BRL), and PSN antibiotics (Gibco BRL)). Before electroporation DNA plasmids pZP-5N/MPL-zalpha11 (example 1) was obtained and was purified using a kit Qiagen Maxi Prep kit (Qiagen) according to the manufacturer's instructions. The BaF3 cells for electroporation were washed once in RPMI medium and then resuspendable in RPMI medium at a density of cells 107cells/ml One ml resuspending BaF3 cells was mixed with 30 μg of DNA plasmid pZP-5N/MPL-zalpha11 and transferred to separate disposable camera for electroporation (Gibco BRL). After a 15 minute incubation at room temperature, the cells were subjected to two consecutive electrical pulses (800 MT/300 V; 1180 MT/300 V), supplied by a device for electroporation (CELL-PORATOR™; Gibco BRL). After 5 minutes of recovery to normal elektrooborudovanie cells was transferred into a 50 ml complete medium and were placed in a thermostat at 15-24 h (37°C, 5% CO2). These cells then Unscrew and resuspendable in 50 ml of complete medium containing Geneticin™ (Gibco) for selection (500 μg/ml G418)in a flask T-162 to highlight S-resistant pool. Pools tra is svirirojana BaF3 cells, hereinafter referred to as cells BaF3/MPL-zalpha11 analyzed on the ability of the transmission signal, as described below.

C. Testing the ability of the transmission signal cells BaF3/MPL-zalpha11 using a dough proliferation with Alamar Blue

Cells BaF3/MPL-zalpha11 Unscrew and washed in complete medium, as described above, but without mlL-3 (hereinafter referred to as "not containing mlL-3 environment"). The cells were turned off and washed 3 times to ensure removal of mlL-3. The cells are then considered in hemocytometer. Cells were planted in 96-well plate at 5000 cells per well in a volume of 100 μl per well using not containing mlL-3 environment.

Cell proliferation BaF3/MPL-zalpha11 was assessed using murine thrombopoietin (mTPO), divorced not containing mlL-3 medium to concentrations of 500 ng/ml, 250 ng/ml, 125 ng/ml, 62 ng/ml, 30 ng/ml, 15 ng/ml to 7.5 ng/ml of 3.75 ng/ml, 1.8 ng/ml, 0.9 ng/ml, 0.5 ng/ml and 0.25 ng/ml 100 ál of diluted tro was added to the cells BaF3/MPL-zalpha11. The total volume of the test is 200 ál. Negative controls were evaluated in parallel using only not containing mlL-3 environment, without adding mTPO. The test plates were incubated at 37°C, 5% CO2within 3 days, after which was added Alamar Blue (Accumed, Chicago, IL) at 20 μl per well. Alamar Blue gives fluorometrically readings based on the number of living cells and is, therefore, a direct measurement of cell proliferation compared to the negative counter is LEM. Tablets are again incubated at 37°C, 5% CO2within 24 hours. Tablets read on a tablet reader Fmax™ (Molecular Devices, Sunnyvale, CA) using SoftMax™ Pro at wavelengths 544 (excitation) and 590 (emission).

The results confirmed the ability of the intracellular signal transmission part zalpha11 receptor as thrombopoietin induced proliferation approximately 10 times higher than background levels, at concentrations mTPO 62 ng/ml and above.

Example 3

Designing expressing vector expressing full-zalpha11

Full zalpha11 receptor was isolated from a plasmid containing cDNA zalpha11 receptor (SEQ ID NO:7), by means of PCR using primers ZC19905 (SEQ ID NO:19) and ZC19906 (SEQ ID NO:20). Reaction conditions were as follows; 95°C for 1 min; 35 cycles at 95°C for 1 minute; 55°C for 1 minute, 72°C for 2 min; then 72°C for 10 minutes; then soaking in 10°C. the PCR product was subjected to electrophoresis on 1% agarose, low melting point (Boehringer Mannheim) and zalpha11 cDNA approximately 1.5 TPN was isolated using a set of for extraction of the Qiaquick gel™ (Qiagen) according to the manufacturer's instructions.

Purified zalpha11 cDNA was digested BamHI (Boehringer Mannheim) and EcoRI (BRL) according to the manufacturer's instructions. All the cleavage product was subjected to electrophoresis on 1% agar is se with a low melting point (Boehringer Mannheim) and purified split zalpha11 fragment with a set of extraction Qiaquick gel according to the manufacturer's instructions. The obtained split zalpha11 fragment was embedded in expressing vector, as described below.

Recipient expressing vector pZP-5N were digested BamHI (Boehringer Mannheim) and EcoRI (BRL) according to the manufacturer's instructions, and purified on a gel as described above. This vector fragment was combined with ratmalana BamHI and EcoRI zalpha11 fragment, selected as described above, in a ligation reaction using T4 ligase (BRL). Ligation was performed by incubation at 15°With during the night. A sample of the ligation was subjected to electroporation in electrocompetent relatively DH10B ElectroMAX™ cells of E. coli (25 μf, 200 Ω, 2.3 In). Transformants were sown on plates with LB+ampicillin and individual colonies were screened using PCR to confirm the sequence zalpha11 using ZC19905 (SEQ ID NO:19) and ZC19906 (SEQ ID NO:20) using the PCR conditions described above.

The confirmation sequence MPL-zalpha11 performed using analysis sequence using the following primers: ZC12700 (SEQ ID NO:10), ZC5020 (SEQ ID NO:11), ZC20114 (SEQ ID NO:21), ZC19459 (SEQ ID NO:22), ZC19954 (SEQ ID NO:23) and ZC20116 (SEQ ID NO:24). The insert was approximately 1,6 TPN and was full.

Example 4

Proliferation on the basis of zalpha11 in F3-test using Alamar Blue A. Construction of BaF3 cells expressing zalpha11 receptor

The BaF3 cells expressing the full-zalpha11 receptor, which was strairway, as in example 2A above, using 30 μg expressing vector zalpha11 described in example 3 above. The BaF3 cells expressing mRNA zalpha11 receptor, were called BaF3/zalpha11. These cells were used for screening the activity of zalpha11 as described below in examples 5 and 6.

Example 5

Screening for zalpha11 ligand from the cell line BaF3/zalpha11 using test proliferation with Alamar Blue

A. Activation of primary splenocytes monkeys to test for the presence of zalpha11 ligand

Splenocyte monkeys stimulated in vitro with obtaining air-conditioned environment for testing for the presence of activity of zalpha11 ligand, as described below. Spleen monkeys were obtained from 8-year-old female monkeys M nesestrian. The spleen was torn to pieces to obtain a suspension of individual cells. Mononuclear cells were isolated using density gradient Ficoll-Paque® PLUS (Pharmacia Biotech, Uppsala, Sweden). Mononuclear cells were sown at 2×106cells/ml in medium RPMI-1640, supplemented with 10% FCS, and activated with 5 ng/ml phorbol-12-myristate-13-acetate (PMA) (Calbiochem, San Diego, CA) and 0.5 mg/ml of Ionomycin™ (Calbiochem) for 48 hours. Supernatant from stimulated cells spleen monkeys used to analyze cell proliferation in BaF3/zalpha11 as described below.

C. Screening for zalpha11 ligand from the cell line BaF3/zalpha11 using test Alamar Blue proliferation/p>

Cells BaF3/zalpha11 Unscrew and washed in not containing mlL-3 environment. The cells were turned off and washed 3 times to ensure removal of mlL-3. The cells are then considered in hemocytometer. Cells were planted in 96-well plate at 5000 cells per well in a volume of 100 μl per well using not containing mlL-3 environment.

Cell proliferation BaF3/zalpha11 was assessed using conditioned medium from activated spleen cells monkeys (see example 5A above). Air-conditioned environment bred not containing mlL-3 medium to concentrations 50%, 25%, 12,5%, 6,25%, 3,125%, 1,5%, 0,75% and the 0.375%. 100 μl of the diluted conditioned medium was added to cells BaF3/zalpha11. The total volume of the test is 200 ál. The test plates were incubated at 37°C, 5% CO2within 3 days, after which was added Alamar Blue (Accumed, Chicago, IL) at 20 μl per well. Tablets are again incubated at 37°C, 5% CO2within 24 hours. Tablets read on a tablet reader Fmax™ (Molecular Devices)as described above (example 2).

The results confirmed the proliferative response of cells BaF3/zalpha11 factor present in the activated air-conditioned environments spleen cells of the monkey. This response, as measured, was approximately 4 times higher than the background at a concentration of 50%. Nitrostilbene cells BaF3 not proliferated in response to this factor, indicating that this factor is pacificism for the Zalpha11 receptor.

C. the primary Source of human cells that is used to allocate Zalpha11 ligand

100 ml of blood was taken from each of the six donors. Blood was extracted using a 10X 10 ml vacuum tubes (vacutainer containing heparin. Blood was pooled from six donors (600 ml), diluted 1:1 in SFR and were separated using Ficoll-Paque® PLUS (Pharmacia Biotech). The output of the selected primary human cells after separation of the gradient ficoll was equal to 1.2×109cells.

Cells suspended in 9.6 ml of MACS buffer (STR, 0.5% EDTA, 2 mm EDTA). 1.6 ml of cell suspension was removed and added to 0.4 ml of microgranules CD3 (Miltenyi Biotec, Auburn, CA). The mixture is incubated for 15 minutes at 4°C. the Cells labeled CD3 granules, washed with 30 ml of MACS buffer and then resuspendable in 2 ml of MACS buffer.

Column VS+ (Miltenyi) was prepared according to the manufacturer's instructions. Then column VS+ was placed in a magnetic field VarioMACS™ (Miltenyi). The column was balanced with 5 ml of MACS buffer. Then the selected primary human cells were applied to this column. D3-negative cells were made to pass through the column. The column is washed with 9 ml (3×3ml) MACS buffer. Then the column was removed from the magnetic field and placed over the tube falcon 15 ml of CD3+ cells was suirable by adding 5 ml of MACS buffer to the column and bound peroxidase cells were washed using a piston (plunger)provided by the manufacturer. And kupirovanie of these cells with D3-magnetic granules, washing and stage speakers VS+ (incubation-elution), described above, was repeated five times. Derived CD3+ fraction from six divisions on the column together. The total yield of CD3+ selected T-cells was equal to 3×108cells.

Sample United CD3+ selected T-cells were taken for staining and sorting cell sorting device with excitation fluorescence (FACS) to assess their purity. CD3+ selected T-cells contained 91% of CD3+ cells.

CD3+ selected T-cells were activated by incubation in RPMI + 5% FCS + PMA 10 ng/ml and inomycin 0.5 μg/ml (Calbiochem) for 13 hours at 37°C. the Supernatant from these activated CD3+ selected T-cells was tested on the activity of zalpha11 ligand, as described below. In addition, activated CD+ selected human cells were used to produce cDNA library as described in example 6 below.

D. Testing supernatant from activated CD3+ selected T-cell person on zalpha11 ligand from the cell line BaF3/zalpha11 and proliferation test using Alamar Blue

Cells BaF3/zalpha11 Unscrew and washed in not containing mlL-3 environment. The cells were turned off and washed 3 times to ensure removal of mlL-3. The cells are then considered in hemocytometer. Cells were planted in 96-well plate at 5000 cells per well in a volume of 100 μl per well using not with the holding mlL-3 environment.

Cell proliferation BaF3/zalpha11 was assessed using conditioned medium from activated CD3+ selected T-cells (see example 5C) above), divorced not containing mlL-3 medium to concentrations 50%, 25%, 12,5%, 6,25%, 3,125%, 1,5%, 0,75% and the 0.375%. 100 μl of the diluted conditioned medium was added to cells BaF3/zalpha11. The total volume of the test is 200 ál. The test plates were incubated and analyzed as described in example 5B.

The results confirmed the proliferative response of cells BaF3/zalpha11 on the factor that is present in the activated air-conditioned environment CD3+ selected T-cells. This response, as measured, was approximately 10 times higher than the background at a concentration of 50%. Nitrostilbene cells BaF3 not proliferated in response to this factor, indicating that this factor is specific for the Zalpha11 receptor. In addition, soluble zalpha11 receptor blocked this proliferative activity in cells BaF3/zalpha11 (see example 11).

Example 6

Cloning of the human zalpha11 ligand from the library CD3+ selected human cells

Screening of cDNA library of primary CD3+ selected activated human cells revealed a dedicated cDNA, which is a new member of the family of cytokines with the structure chetyrekhmernogo beam. This cDNA encodes zalpha11 ligand. This cDNA was identified screenin the ohms on the activity of zalpha11 ligand using zalpha11 receptor.

A. Vector for constructing CD3+ selected library

Vector for constructing CD3+ selected library plasmid was pZP7NX. Vector pZP7NX designed as follows. Coding region for selective marker DHFR in the vector pZP7 was removed by cleavage of DNA with restriction enzymes (restrictable) Ncol and > PST (Boehringer Mannheim). Cleaved DNA was subjected to electrophoresis on 1% agarose gel, cut out and purified from the gel using a kit for the extraction of Qiaquick gel™ Gel Extraction Kit (Qiagen) according to the manufacturer's instructions. The DNA fragment encoding the region of selective marker Zeocin, amplified using PCR with primers ZC13946 (SEQ ID NO:25) and ZC13945 (SEQ ID NO:26) and pZeoSV2(+) as the matrix. In the primer ZC13946 (SEQ ID NO:25) had an additional restriction sites > PST and Bell, and additional sites Ncol and Sful had in the primer ZC13945 (SEQ ID NO:26). The PCR fragment was cut with restrictase > PST and Ncol and cloned in the vector pZP7 obtained by splitting the same two enzymes, followed by purification from the gel. This vector was named pZP7Z. Then coding region Zeocin was removed by cleavage of the DNA vector pZP7Z restrictase Bell and Sful. Cleaved DNA was subjected to electrophoresis on 1% agarose gel, cut out and purified from the gel and then ligated with a DNA fragment encoding the neomycin district, carved out of a vector pZem228 (deponirawe the tion in the American type culture collection (ATSS), Manassas, VA; ATCC Deposit No. 69446) using the same restricted (Bell and Sful).

This new vector was named pZP7N, in which the coding region for selective marker DHFR was replaced with the coding region for selective marker of neomycin vector pZem228. Fragment-filler containing an Xhol site, added to pZP7N to create a vector, suitable for highly efficient directional cloning of cDNA; this new vector was named pZP7NX. To prepare the vector for cDNA, 20 µg pZP7NX were digested with 20 units of EcoRI (Life Technologies, Gaithersburg, MD) and 20 units Xhol (Boehringer Mannheim, Indianapolis, IN) for 5 hours at 37°then 68°C for 15 minutes. The cleavage product was then subjected to electrophoresis on 0.8% low-melting agarose gel HTAE to separate the filler from the vector. Band vector cut and split "beta agarti" (New England Biolabs, Beverly, MA) according to the manufacturer's recommendations. After precipitation with ethanol split vector resuspendable in water up to 45 ng/ml in preparation for ligation of CD3+ selected cDNA library described below.

C. obtaining a cDNA library of primary CD3+ selected activated human cells

Approximately 1.5×108primary CD3+ selected human cells stimulated with ionomycin/PMA, were isolated by centrifugation after culturing at 37°C for 13 hours (note the R 5C). Total RNA was isolated from sediment of cells using the set RNeasy Midi" from Qiagen, Inc. (Valencia, CA). mRNA was isolated from 225 µg of total RNA using a kit for purification of mRNA MPG mRNA purification kit" from CPG Inc. (Lincoln Park, NJ). 3.4 micrograms of mRNA was isolated and converted into double-stranded cDNA using the following procedure.

cDNA first chain of stimulated CD3+ selected human cells synthesized as follows. Nine μl of oligo d(T)-selected poly(A) CD3+ RNA at a concentration of 0.34 μg/μl and 1.0 μl of 1 μg/μl of primer first circuit ZC18698 (SEQ ID NO:27)containing an Xhol restriction site, were mixed and heated at 65°C for 4 minutes and cooled on ice. Synthesis of cDNA the first chain was initiated by addition of 9 μl of the buffer for the first circuit (5x SUPERSCRIPT ®-buffer (Life Technologies), 4 μl of 100 mm dithiothreitol and 2 μl of a solution of deoxynucleotidase containing 10 mm each of dATP, dGTP, dTTP and 5-methyl-asters (Pharmacia Biotech, Inc.), to a mixture of RNA primer. The reaction mixture was incubated at 45°C for 4 minutes followed by the addition of 8 μl of 200 U/μl SUPERSCRIPT®, Mcasa H - reverse transcriptase (Life Technologies). The reaction was incubated at 45°C for 45 minutes, followed by lowering the temperature of incubation on 1°every 2 minutes up to 50°and the reaction was maintained at 50° within 10 minutes. To denature any secondary structure and additional permission is sustained fashion lengthening this cDNA reaction was then heated to 70° C for 2 minutes, then reduced the temperature to 55°C for 4 minutes, after which was added 2 μl OF (reverse transcriptase) SUPERSCRIPT® and incubated additionally for 15 minutes, followed by lowering the temperature to 70°1 minute/1°C. not Included nucleotides were removed from the cDNA by precipitation twice in the presence of 2 μg glycogen carrier, 2.0 M ammonium acetate and 2.5 volume of ethanol followed by rinsing with 100 ál of 70% ethanol. This cDNA resuspendable in 98 ál of water for use in the synthesis of the second chain.

The synthesis of the second chain was performed on cDNA first chain in the conditions which have promotional priming the first circuit of the second synthesis circuit, which led to the formation of DNA hairpins. The reaction of the second circuit contained 98 ál of cDNA first circuit, 30 ál of 5x buffer polymerase I (100 mm Tris-HCI, pH 7.5, 500 mm KCI, 25 mm MgCl250 mm (NH4)2SO4, 2 MCP 100 mm dithiothreitol, 6 μl of a solution containing 10 mm each of deoxynucleotidase, 5 μl 5 mm b-NAD, 1 μl of 3 U/μl DNA ligase E. coli (New England Biolabs Inc.) and 4 ál 10 U/ál DNA polymerase 1 E. coli (New England Biolabs Inc.). The reaction was assembled at room temperature and incubated at room temperature for 2 minutes, followed by addition of 4 μl of 3.8 U/μl RNase H (Life Technologies). The reaction was incubated at 15°C for two hours with a subsequent 15-minute inkubirovanie the m at room temperature. To the reaction was added 10 μl of 1 M TRIS pH 7.4 and were extracted twice with a mixture of phenol-chloroform and once with chloroform, then the organic phase was back extracted with 50 μl of TE (10 mm TRIS pH 7.4, 1 mm EDTA), combined with other water phase and precipitated with ethanol in the presence of 0.3 M sodium acetate. The precipitate was washed with 100 ál of 70% ethanol, dried in air and resuspendable in 40 μl of water.

Single-stranded DNA hairpin structures were digested with nuclease bean Golden. The reaction mixture contained 40 μl of the second chain cDNA, 5 ál of 10x buffer for nucleases bean Golden (Life Technologies), 5 ál of nuclease bean Golden (Pharmacia Biotech, Corp.), diluted to 1 U/ál in 1x buffer for nucleases bean Golden. The reaction was incubated at 37°C for 45 minutes. The reaction was stopped by adding 10 II 1 M Tris-HCl, pH 7.4, followed by successive extraction with a mixture of phenol/chloroform and chloroform as described above. After extraction cDNA precipitated with ethanol in the presence of 0.3 M sodium acetate. The precipitate was washed with 100 ál of 70% ethanol, dried in air and resuspendable in 38 μl of water.

Resuspending cDNA was a small mistake at the ends of the DNA polymerase, T4. cDNA, which resuspendable in 38 μl of water was mixed with 12 µl of 5x buffer for DNA polymerase T4 (250 mm Tris-HCl, pH 8.0, 250 mm KCL, 25 mm MgCl2), 2 μl of 0.1 M dithiothreitol, 6 μl of a solution containing 10 mm each is about from deoxynucleotidase, and 2 ál 1 U/ál DNA polymerase T4 (Boehringer Mannheim, Corp.). After incubation for 45 minutes at 15°the reaction was stopped by adding 30 μl of TE followed by successive extraction with a mixture of phenol/chloroform and chloroform and back was extracted with 20 μl of TE, as described above. This DNA is precipitated with ethanol in the presence of 2 μl of medium Pellet Paint™ (paint sludge) (Novagen) and 0.3 M sodium acetate and resuspendable in 11 μl of water.

Adapters are ligated EcoRI at the 5'ends of the cDNA described above, to facilitate cloning in expressing vector. 11 μl of cDNA and 4 µl of 65 pmol/ál provospalitionah adaptor Eco R1 (Pharmacia Biotech Corp.) was mixed with 5 ál of 5x ligase buffer (Life Technologies), 2 μl 10 mm ATP and 3 μl of 1 U/ál DNA T4 ligase (Life Technologies), 1 µl 10x buffer for ligation (Promega, Corp.), 9 μl of water. Another 1x dilution buffer was made to prevent deposition of the paint Pellet Paint. The reaction is incubated for 9 hours on a water bath with a temperature change from 10 to 22°C for 9 hours, then kept for 45 minutes at 25°C. the Reaction was stopped by incubation at 68°C for 15 minutes.

To facilitate directional cloning of cDNA in expressing vector cDNA was digested with Xhol obtaining cDNA with 5'-EcoRl-sticky end and the 3'Xhol-sticky end. The Xhol restriction site at the 3'end of this DNA was pre-entered with the COI is whether the primer ZC18698 (SEQ ID NO:27). Splitting restrictase was performed in a reaction mixture containing 35 μl of the mixture for ligation described above, 6 ál of 10x H buffer (Boehringer Mannheim Corp.), 1 ál of 2 mg/ml BSA (Biolabs Corp.), 17 μl of water and 1.0 μl of 40 U/ál Xhol (Boehringer Mannheim). Cleavage was carried out at 37°C for 1 hour. The reaction was stopped by incubation at 68°C for 15 minutes followed by ethanol precipitation, washing and drying, as described above, and resuspending in 30 µl of water.

Resuspending cDNA was heated to 65°C for 5 minutes and cooled on ice, was added 4 μl of 5x dye to be applied on the gel (Research Genetics Corp.) CDNA was applied at the 1X TAE to 0.8% low-melting agarose (SEA PLAQUE GTG™ low melt agarose; FMC Corp.) and subjected to electrophoresis. Impurity adapters and cDNA length below 0.6 TPN cut out of the gel. The electrodes were changed places, molten agarose dobavlyali to fill the wells, the buffer was replaced and cDNA was subjected to electrophoresis to its concentration near the beginning of the track. The area of the gel containing concentrated cDNA was cut out and placed in microporous tube, and the agarose was melted by heating to 65°C for 15 minutes. After equilibration of the sample to 45°added 2 ál 1 U/ál beta-agarose I (Biolabs Inc.) and the mixture is incubated for 90 minutes at 45°for cleavage of agarose. After incubation 1/10 volume 3 M Na-acetate buffer add the Yali in the sample and the mixture incubated on ice for 15 minutes. The sample was centrifuged at 14000 × g for 15 minutes at room temperature to remove undigested agarose, cDNA precipitated with ethanol, washed in 70% ethanol, dried in air and resuspendable in 40 μl of water.

To determine the optimal relationship of cDNA to vector several legirovanii assembled and electroporative. Briefly, 2 μl of 5x buffer for T4 ligase (Life Technologies), 1 μl 10 mm ATP. 1 µl pZP7NX, split EcoRl-Xhol, 1 II DNA T4 ligase diluted to 0.25 U/μl (Life Technologies) water to 10 μl and 0.5, 1, 2 or 3 μl of cDNA were mixed in 4 separate legirovanyh, incubated at 22°C for 4 hours, 68°C for 20 minutes, precipitated with a mixture of sodium acetate-ethanol, washed, dried and resuspendable 10 II. One microliter of each ligation was electroporative in 40 μl of ElectroMAX DH10b™-electrocompetent bacteria (Life Technologies) using a 0.1 cm cuvette (Biorad) and a Genepulser, the controller pulses™ (Biorad)set at 2.5 kV, 251 F., 200 Ohms. These cells immediately resuspendable in 1 ml of SOC broth (Manniatis et al., supra) followed by the addition of 500 H of a mixture of 50% glycerol-SOC as a preservative. These "source glycerin solutions were frozen in the form of multiple aliquot at -70°C. an Aliquot of each thawed and were sown serially to plates with LB-agar supplemented with ampicillin at 100 µg/ml in the Number of colonies showed that the optimal ratio of CD3 cDNA to vector pZP7NX was 1 mm to 45 ng; this ligation was given 4.5 million primary clones.

For the purpose of screening the library using the test of proliferation on the basis of BaF3-zalpha11 (example 5) glycerin source solutions, described above, was diluted in liquid culture 100 or 250 clones per pool in microtiter tablets with deep holes were grown for 24 hours at 37°With shaking and the plasmids were isolated using the kit Qiagen according to the manufacturer's instructions. This DNA was then transfusional cells KSS mreb was kondicionirovanie 72 hours and were placed on the cells 5K BaF3-zalpha11 and kept for 72 hours, after which proliferation was assessed using a fluorescent assay with Alamar blue (example 5B and example 2B).

For the purpose of screening the library by cloning with secretory trap complex amplificatory the form of a library required for transfection of cells COS-7. About 4.8 million clones were sown at 110 cups 15 cm LB-agar supplemented with 100 µg/ml ampicillin, 10 μg/ml methicillin. After growing cups over night at 37°bacteria were collected by scratching and besieged. Plasmid DNA was extracted from the besieged bacteria using Nucleobond-giga™ (Clontech) according to the manufacturer's instructions. Then, this plasmid was used for transfection of cells COS-7 (ATCC No. CRL 1651) on slides and subjected is whether the screening method secretory trap described below (example 12).

Example 7

Expression cloning of zalpha11 ligand person

Glycerin source solutions from the library of activated CD3+ selected human cells (example 6) was added to SuperBroth II™ (Becton Dickinson, Cockeysville, MD) +0.1 mg/ml ampicillin (amp) at a concentration of 250 cells per 800 microliters. E. coli was given to balanced within 24 hours at room temperature. During inoculation 400 microlitres were sown on plates with LB + amp to determine the actual title of inoculation. After 24 hours, a Cup of thought and then a final concentration of SuperBroth II™ + E. coli brought such a way that the final concentration was 250 cells of 1.2 ml was Inoculable 3 times in 2 liters of obtaining a total of 6 liters. Then this environment were sown in 96-well blocks with deep holes (Qiagen). Sowing was performed on 8-channel bottling device Q-Fill2™ (Genetix, Christchurch, Dorset, UK). E. coli were grown overnight at 37°C With shaking at 250 rpm on a multi-shaker New Brunswick Scientific Innova 4900 multi-tier environment shaker. E. coli was unscrewed from the solution at 3000 rpm using a centrifuge Beckman GS-6KR. Precipitation of E. coli were frozen at -20°With or used fresh before getting minipreparation plasmid DNA. Each residue contains approximately 250 cDNA clones from the library CD3+ selected human cells.

Then these poly cDNA clones were prepared using the kit QIAprep ™ 96 Turbo Miniprep (Qiagen). Plasmid DNA was suirable using 125 μl of TE (10 mm Tris pH 8, 1 mm EDTA). Then, this plasmid DNA was used for transfection of cells KSS.

Transfection of KSS

Cells KSS were sown in 96-well plates to tissue culture at a density of 12,000 cells per well in a volume of 100 μl per well. The culture medium was DMEM (Gibco BRL), 5% inactivated by heating fetal calf serum, 2 mm L-glutamine (Gibco BRL), 1x PSN (Gibco BRL), 1 mm sodium pyruvate (Gibco BRL).

The next day cells KSS were washed once with 100 µl of the SFA. SFA is a serum-free environment, which is DMEM/F12 (Gibco BRL), 2 mm GlutaMax™ (Gibco BRL), 1 mm sodium pyruvate, 10 μg/ml transferrin, 5 μg/ml insulin, 10 μg/ml of fetuin, 2 µg/ml selenium, 25 mm HEPES (Gibco BRL), 100 μm non-essential amino acids (Gibco BRL).

The mixture of DNA/Lipofectamine™ prepared as follows: 2,2 ál Lipofectamine™ (Gibco BRL) combined with 102,8 ál of SFA at room temperature; then add approximately 5 ál of plasmid DNA (200 ng/ál) to Lipofectamine™/RA with the formation of a mixture of DNA/Lipofectamine™, which is incubated at room temperature for 30 minutes. SFA was removed from the cells KSS and the cells were incubated with 50 μl of a mixture of DNA/Lipofectamine™ for 5 hours at 37°With 5% CO2. Fifty μl of a mixture of DNA/Lipofectamine™ was added to each of the two holes CL is the current KSS so transfection was performed in duplicate.

After incubation cells KSS with a mixture of DNA/Lipofectamine™ for 5 hours a mixture of DNA/Lipofectamine™ was removed and added to 100 μl of culture medium. Cells were incubated overnight, the medium was removed and replaced with 100 μl of culture medium. After culturing the cells for 72 hours, the conditioned medium was removed, frozen at -80°C for at least 20 minutes, thawed, and then 50 μl were analyzed in the test proliferation zalpha11/BaF3 described in example 2 and example 5, to identify pools of 250 clones with activity of the ligand.

Thirty-five 96-well plates were subjected to screening in a single analysis. This represented approximately 250 cDNA per well or just 840000 cDNA. Of them air-conditioned environment of the 54 holes (representing 250 cDNA per well) were tested as positive in the test of proliferation. Air-conditioned environment of these positive pools re-tested in the second test (secretory trap) with soluble receptor or without it (see example 12). Soluble zalpha11CEE-receptor (example 10A) was used at a final concentration of about 1 μg/ml). For all 54 of positive clones essentially all the activity was kind of balanced out by the addition of soluble zalpha11 receptor, suggesting that these pools contained zalpha11 cDNA-League is Yes. Four positive clones were selected for colony collapse and separate cDNA, which encodes the zalpha11 ligand. These clones were S, 46G11, N and A.

For each of these 4 pools, 1 µl of DNA was used to transform ElectroMax™ DH10B cells (Gibco BRL) spectraforce. Transformants were sown on LB + amp (100 μg/ml) + methicillin (10 μg/ml) to obtain isolated colonies. For each Elektroprivreda pool 960 individual colonies were viscerale toothpick in ten 96-well plates containing 1.2 ml SuperBroth II™ per well. These tablets were numbered No. 1-10 for each of the broken pools (S, 46G11, 401-112 and A). They were cultured overnight and plasmid DNA was obtained in the form of minipreparation, as described above. For 46G11, N and A plasmid DNA from cups to the destroyed colonies were transfusional cells KSS, as described above.

For S used Protocol "fast tracking" to accelerate the identification of cDNA zalpha11 ligand. Cells KSS was transfusional plasmid DNA from cups with the destruction of the colonies, as described above, the mixture of DNA/Lipofectamine™ was removed after 5 hours of incubation and added to the culture medium. Because transfection was carried out in two replications, the culture medium was collected the next day after 24 hours from one of transfected cups and collected the next day posle hours from another transtitional Cup. Air-conditioned environment 24 hours were analyzed, as described above, on the activity of zalpha11 ligand using a dough proliferation, as described above.

Plasmid DNA was combined from the cups to the destroyed colonies S No. 1-4 and analyzed for binding to soluble zalpha11 receptor with its ligand Protocol "secretory trap (see example 12 below). Eight positive clones were identified from 384 sequences in General. The results of the test confirmed the proliferation activity of zalpha11 ligand, and they correlated with the results of the test secretory trap (see example 12). In parallel, the plasmid DNA obtained in the form of minipreparation of cups No. 1-4 destroyed pool S, sequenced to determine the DNA sequence of each of the 384 clones.

Several clones were identified as positive in the tests proliferation and secretory trap, also sequenced using the following primers: ZC14063 (SEQ ID NO:28), ZC7764a (SEQ ID NO:38), ZC7764b (SEQ ID NO:39), ZC22034 (SEQ ID NO:40) and ZC22035 (SEQ ID NO:41). Polynucleotide sequence zalpha11 ligand was full (SEQ ID NO:1), and its corresponding amino acid sequence shown in (SEQ ID NO:2).

Example 8

Designing expressing vectors mammals that Express zalpha11 soluble receptors: zalpha11CEE, zalpha11CFLG, zalpha11CHIS and zalpha11-Fc4

<> A. Designing expressing vector zalpha11 mammals, containing zalpha11CEE. ZaIphaUCFLG, zalpha11CHIS

Received expressing vector for expression of the soluble extracellular domain of the zalpha11 polypeptide, pC4zalpha11CEE, and this design is intended for the expression of zalpha11 polypeptide containing the predicted initiating methionine and truncated near the predicted transmembrane domain with C-terminal Glu-Glu tag (SEQ ID NO:29).

PCR-generated DNA fragment zalpha11 700 BP was created using ZC19931 (SEQ ID NO:30) and ZC19932 (SEQ ID NO:31) as PCR primers to add restriction sites Asp718 and BamHI. The plasmids containing cDNA zalpha11 receptor (SEQ ID NO:7), was used as matrix. PCR amplification of zalpha11 fragment was carried out as follows: twenty-five cycles at 94°C for 0.5 minutes; five cycles at 94°C for 10 seconds, 50°C for 30 seconds, 68°C for 45 seconds; then keeping at 4°C. the Reaction was purified by extraction with a mixture of chloroform/phenol and precipitation with isopropanol and uncoupled Asp718 and BamHI (Gibco BRL) according to the manufacturer's instructions. The band of the predicted size, 700 gel, visualized by electrophoresis on 1% agarose gel, cut out and the DNA was purified using a purification system Qiaexll™ (Qiagen) according to the manufacturer's instructions.

Cut DNA su is cloned into a plasmid rsee, which was cut BamHI and Asp718. Expressing the vector pC4zalpha11CEE uses a signal peptide native zalpha11 and attaches Glu-Glu tag (SEQ ID NO:29) to the C-end of the polynucleotide sequence that encodes a polypeptide zalpha11. Plasmid rsee is expressing vector mammals, containing the expression cassette with the mouse promoter metallothionein-1, multiple restriction sites for installation of coding sequences, a stop codon and a terminator of human growth hormone. This plasmid is also the beginning of replication of E. coli, the expression unit of breeding markers mammals, with the promoter, enhancer and early SV40 replication, a DHFR gene, and the SV40 terminator.

Approximately 30 ng restriction split zalpha11-insert and about 12 ng of the split vector ligated overnight at 16°C. One microliter of each ligation reaction were independently electroporative in DH10B competent cells (Gibco BRL, Gaithersburg, MD) according to the manufacturer's instructions, and were sown on LB-tablets containing 50 mg/ml ampicillin, and incubated overnight. Colonies were screened using restriction analysis of DNA obtained from 2 ml liquid cultures of individual colonies. The sequence of the inserts of positive clones were confirmed by sequence analysis. Large-scale p is torching plasmids was performed using a kit QIAGEN® Maxi prep kit (Qiagen) according to the manufacturer's instructions.

The same process was used to obtain the soluble zalpha11 receptor with C-terminal His-tag consisting of 6 His residues in a row, and C-terminal FLAG-tag (SEQ ID NO:37), zalpha11CFLAG. To construct these structures the above vector has either HIS, or P1-JSC®tag instead of a glu-glu tag (SEQ ID NO:29).

C. Expression design mammals soluble zalpha11 receptor. zalpha11-Fc4

The expression plasmid containing the entire polynucleotide or part thereof, encoding zalpha11, designed by homologous recombination. The extracellular domain zalpha11 receptor was merged with Fc region derived from IgG person, he was named "Fc4" (SEQ ID NO:33) and contains a mutation, so he is not already associated with Fc-receptor. Using the PCR fragment was isolated zalpha11 cDNA, which includes a polynucleotide sequence of the extracellular domain of the zalpha11 receptor. Two primers were used to obtain this zalpha11 fragment: (1) each of the primers for PCR comprises from 5' to 3' end: 40 BP of flanking sequence vector (5' from inserted) and 17 BP corresponding to the 5'-end of the extracellular domain zalpha11 (SEQ ID NO:32); and (2) 40 BP 5'-end of the polynucleotide sequence Fc4 (SEQ ID NO:33) and 17 BP corresponding to the 3'-end of the extracellular domain zalpha11 (SEQ ID NO:34). The Fc4 fragment to merge with zalpha11 of Generalov is whether using PCR in a similar manner. Two primers in obtaining the Fc4 fragment were: (1) 5'-primer, consisting of a 40 BP sequence from the 3'end of the extracellular domain zalpha11 and 17 BP of 5'end of Fc4 (SEQ ID NO:35), and (2) 3'-primer, consisting of 40 BP of vector sequence (3' from inserted) and 17 BP of the 3'end of Fc4 (SEQ ID NO:36).

PCR amplification of each of the reactions described above, was carried out as follows: one cycle at 94°C for 2 minutes; twenty-five cycles at 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 min; one cycle at 72°C for 5 minutes; followed by keeping at 4°C. Ten μl of the 100 μl PCR reaction was subjected to electrophoresis on of 0.8% LMP-agarose gel (Seaplaque GTG) with 1 × TBE-buffer for analysis. The remaining 90 μl of PCR reaction was besieged by adding 5 ál of 1 M NaCl and 250 μl of absolute ethanol. Used expressing vector was obtained from plasmid pCZR199 obtained from pZP9 (ATCC Deposit No. 98668) and cut Smal (BRL). Expressing vector was produced from plasmid pCZR199 and represents expressing vector mammals, containing the expression cassette with immediate early CMV promoter, a consensus intron of variable regions of the heavy chain locus of the mouse immunoglobulin, multiple restriction sites for installation of coding sequences, a stop codon and a terminator growth hormone human the and. This expressing vector is also the beginning of replication of E. coli, the expression unit of breeding markers mammals, with the promoter, enhancer and early SV40 replication, a DHFR gene, and the SV40 terminator. This used expressing vector was designed from pCZR199 replacing the promoter of metallothionein immediate early CMV promoter.

100 μl of competent yeast cells (S. cerevisiae) were combined with 10 µl containing approximately 1 μg of each of the inserts zalpha11 and Fc4 and 100 ng of Smal split (BRL) expressing vector, and transferred into a cuvette for electroporation see 0,2 Mix the yeast/DNA was subjected to electric shocks at 0.75 kV (5 kV/cm), "undefined" number of Ohms, 25 μf. To each cuvette was added 600 μl of 1.2 M sorbitol and the yeast sown in two aliquot of 300 μl on two cups of URA-D and incubated at 30°C.

After approximately 48 hours Ura+ yeast transformants from a single Cup resuspendable in 1 ml of H2O and Unscrew the fast sedimentation of yeast cells. Cellular precipitate resuspendable in 1 ml lisanova buffer (2% Triton X-100, 1% LTOs, 100 mm NaCl, 10 mm Tris, pH 8.0, 1 mm EDTA). 500 ál lizinoj mixture was added to the Eppendorf tube containing 300 μl acid washed glass granules and 200 ál of a mixture of phenol/chloroform was shaken on a vortex two or three times at intervals of 1 min, followed by 5-minute loosening in C is trifuge Eppendorf at maximum speed. Three hundred microlitres the aqueous phase was transferred into a fresh tube and DNA was besieged by 600 ál of ethanol (EtOH), followed by centrifugation for 10 minutes at 4°C. the precipitated DNA resuspendable in 100 μl of N2O.

Transformation electrocompetent the cells of E. coli (DH10B, Gibco BRL) carried out with 0.5-2 ml yeast DNA and 40 μl of DH10B cells. Cells were subjected to electrical pulses at 2.0 kV, 25 MF and 400 Ohms. After electroporation, 1 ml SOC (2% Bacto™ tripton (Difco, Detroit, Ml), 0.5% of yeast extract (Difco), 10 mm NaCl, 2.5 mm KCI, 10 mm MgCb, 10 mm MgSO4, 20 mm glucose) were sown in the aliquot 250 ál four LB AMR Cup (LB-broth (Lennox), 1.8% Baktagir (Difco), 100 mg/l ampicillin).

Individual clones carrying the correct expression construct for zalpha11-Fc4 identified restriction cleavage to confirm the presence of insert zalpha11-Fc4 and to confirm that the various DNA sequences have been correctly connected to each other. Insert positive clones were subjected to sequence analysis. When obtaining large-scale plasmid DNA secrete using a set of QIAGEN Maxi kit (Qiagen) according to the manufacturer's instructions.

Example 9

Transfection and expression of polypeptides soluble zalpha11 receptor

A. Expression in mammalian soluble zalpha11 receptor zalpha11CEE. Zalpha11CFLG and zalpha11CHIS

Cells KSS 570 (ATSS No. CRL-10314), p is SAG 27, sown in the Cup at 1.2×106cells per well (6-hole Cup) in 800 μl of serum-free (BS) medium DMEM (DMEM, Gibco/BRL High Glucose), (GIBCO BRL, Gaithersburg, MD). These cells were transfusional expression plasmids containing zalpha11CEE, zalpha11CFLG or zalpha11CHIS described above (see example 8), using Lipofectin™ (Gibco BRL), in serum free (BS) DMEM. Three micrograms zalpha11CEE, zalpha11CFLG or zalpha11CHIS separately bred in test tubes 1.5 ml to a total volume of 100 μl BS-DMEM. In a separate test tubes 15 ál Lipofectin™ (Gibco BRL) was mixed with 100 ál of BS-DMEM. The mixture Lipofection™ incubated at room temperature for 30-45 minutes, then add the mixture of DNA and allowed to incubated for approximately 10-15 minutes at room temperature.

All the mixture of DNA:Lipofectin™ was added to the seeded cells and uniformly distributed on them. The cups were incubated at 37°for about five hours, then transferred into individual cups MAXI 150 mm in a final volume of 30 ml DMEM/15% fetal calf serum (FCS) (Hyclone, Logan, UT). The cups were incubated at 37°C, 5% CO2during the night, and the mixture of DNA:Lipofectin™ was replaced with fresh selective medium (5% FCS/DMEM with 1 μm methotrexate (MTX) on the next day.

At approximately 10-12 days after transfection cups washed with 10 ml of BS-DMEM. The wash medium was aspirated and replaced 7,25 ml DMEM. Then a sterile Teflon mesh (Spectrum Medical Industries, Los Angeles, CA), pre-soaked in BS-DMEM, were placed on the colonies of clonal cells. Then this grid was placed sterile nitrocellulose filter, pre-soaked in BS-DMEM. Label for orientation on the nitrocellulose were transferred to the Cup with the culture. Then these cups were incubated for 5-6 hours in a thermostat at 37°C, 5% CO2.

After incubation the filters/mesh was removed and the medium was aspirated and replaced with medium with 5% FCS/DMEM with 1 μm MTX. Then the filters were blocked in 10% nonfat dry milk/buffer And Western (Western A: 50 mm Tris, pH 7.4, 5 mm EDTA, 0.05% of NP-40, 150 mm NaCl and 0.25% gelatin) for 15 minutes at room temperature on a rotating shaker. Then the filters were incubated with the antibody against Glu-Glu, antibody against FLAG® or conjugates of anti-His-HRP (horseradish peroxidase), respectively 2.5% nonfat dry milk/buffer Western And for one hour at room temperature on a rotating shaker. Then the filters were washed three times at room temperature with buffer Western And within 5-10 minutes per wash. The filters showed the ultra reagent ECL (Amersham Corp., Arlington Heights, IL) according to the manufacturer's instructions and visualized on the device for acquiring images Lumi-lmager (Roche Corp.).

Positive expressing clonal colonies mechanical Vick is fucked in 12-hole Cup in one ml of 5% FCS/DMEM with 5 μm MTX, then grew to confluently. Then samples of conditioned media were tested for the levels of expression by means of electrophoresis in LTO-page and Western analysis. The three most highly expressing clone for each construct was viscerale; two of the three froze in reserve, and one was developed for testing for Mycoplasma and large-scale planting in the cell factory.

C. Expression of soluble zalpha11 receptor zalpha11-Fc4 in mammalian cells

Cells KSS 570 (ATSS No. CRL-10314) were sown in Cup 10 cm tissue culture and gave them to grow to approximately 50-70% of confluently over night at 37°C, 5% CO2in the DMEM/FCS (DMEM, Gibco/BRL High Glucose, (GIBCO BRL, Gaithersburg, MD), 5% fetal calf serum (Hyclone, Logan, UT), 1 mm L-glutamine (JRH Biosciences, Lenexa, KS), 1 mm sodium pyruvate (Gibco BRL). Then the cells were transfusional a plasmid containing zalpha11-Fc4 (see example 8), using Lipofectamine™ (Gibco BRL), in serum free (BS) medium (DMEM, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml of fetuin, 1% L-glutamine and 1% sodium pyruvate). The plasmids containing zalpha11-Fc4, bred in test tubes 15 ml to a total final volume of 640 μl BS environment. 35 ml of Lipofectamine™ (Gibco BRL) was mixed with 605 μl of BS. This mixture with Lipofectamine™ was added to the mixture of DNA and allowed to incubated for approximately 30 minutes at room temperature the E. Five milliliters BS-medium was added to the mixture of DNA: Lipofectamine™. Cells were washed once with 5 ml of BS-environment, was aspirated and added to the mixture of DNA: Lipofectamine™. Cells were incubated at 37°C for five hours, then in each Cup was added to 6.4 ml of medium DMEM/10% FCS, 1% PSN. The cups were incubated at 37°during the night and the mixture of DNA: Lipofectamine™ was replaced with fresh medium with 5% FCS/DMEM for the next day. At day 2 after transfection cells were separated in a selective medium (DMEM/FCS, as described above, with the addition of 1 mm methotrexate (Sigma Chemical Co., St. Louis, MO)) in the cups 150 mm at a dilution of 1:1, 1:20 and 1:50. Environment on these cells was replaced with fresh selective medium at day 5 after transfection. Around day 10 after transfection two cultural cups 150 mm resistant to methotrexate colonies from each transfection were trypsinization and cells were combined and were sown in the flask T-162 and carried in large-scale culture.

Example 10

Purification of a soluble zalpha11 receptor from cells KSS 570

A. Purification of the polypeptide zalpha11CEE from KSS 570

Unless otherwise noted, all operations were carried out at 4°C. the Following procedure was used for purification of the zalpha11 polypeptide containing C-terminal tag, Glu-Glu (EE). Thirty liters of conditioned medium from the cell factory was concentrated to 1.6 liters by means of helical Cartagena S10Y3 on ProFlux A30. The solution proteasome inhibitor was added to the concentrated 1.6 liter air-conditioned environment of the cell factory from transfected cells KSS 570 (see example 9) to final concentrations of 2.5 mm ethylendiaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis, MO), 0,003 mm leupeptin (Boehringer Mannheim, Indianapolis, IN), 0.001 mm of pepstatin (Boehringer Mannheim) and 0.4 mm Pefabloc (Boehringer Mannheim). Samples were removed for analysis and the total volume was frozen at -80°before cleaning. The concentration of the target protein concentrated conditioned medium of cell factories was determined by electrophoresis in LTO-SDS page and Western blot analysis with antibodies against IT, conjugated with HRP (horseradish peroxidase).

100 ml of substrate for speakers anti-EE-G-Sepharose (prepared as described below) was poured in a glass column, Waters AP-5, 5 cm × 10 cm Column was Packed flowing and balanced on a BioCad Sprint (PerSeptive BioSystems, Framingham, MA) phosphate buffered saline (SFR) pH 7.4. Concentrated conditioned medium of cell factories thawed, sterile filtered with a filter of 0.2 microns, the pH was brought to 7.4, and then applied to the column during the night with a flow rate of 1 ml/min. and the Column washed with 10 column volumes (KO) taputeranga phosphate salt solution (SFR, pH 7.4), then suirable with the flow of eluent through the tube 200 ml SFR (pH 6.0), containing the his of 0.5 mg/ml IT-peptide (Anaspec, San Jose, CA), at a speed of DC 5 ml/min Used-peptide has the sequence EYMPME (SEQ ID NO:29). The column washed with 10 column volumes (KO) SFR, then suirable 5 column volumes of 0.2 M glycine, pH 3.0. pH eluruumi glycine column was brought to 7.0 with 2 column volumes of 5X STR, then uravnovesheni in SFR (pH 7.4). Fractions of 5 ml were collected throughout elsinoe chromatography and absorption at 280 and 215 nm were subjected to monitoring; passing through the column, and wash pools well maintained and analyzed. The fraction of the peak elution HER-polypeptide was analyzed for the target protein electrophoresis in LTO-page with silver staining and Western blotting with conjugated antibody anti-EE-HRP. Interest fraction elution of the polypeptide were pooled and concentrated from 60 to 5.0 ml using a centrifugal concentrator with a membrane, cut-off molecular weight of 10,000 daltons (Millipore, Bedford, MA)according to the manufacturer's instructions.

For the Department zalpha11CEE from other suicidality concentrated proteins fused fraction elution of the polypeptide was subjected to purification on a POROS HQ-50 (strong anion exchange column from PerSeptive BioSystems, Framingham, MA) at pH 8.0. Column 1,0×6.0 cm was flooded and flowing Packed on a BioCad Sprint. The column was loaded by the counterion and then balanced in 20 mm Tris pH 8.0 (Tris(hydroxyethylaminomethyl)). About what u were diluted 1:13 (to reduce ionic strength SFR), then was applied on the column POROS HQ at the speed of a current of 5 ml/min. and the Column washed with 10 column volumes (KO) 20 mm Tris pH 8.0, was then suirable 40 column volume gradient of 20 mm Tris/1 M sodium chloride (NaCl) at a speed of DC 10 ml/min Fractions of 1.5 ml were collected throughout chromatography and absorption at 280 and 215 nm were subjected to monitoring. The fraction of the peak elution were analyzed by electrophoresis in LTO-page with silver staining. Interest fractions were combined and concentrated to 1.5-2 ml using a centrifugal concentrator with a membrane, cut-off molecular weight of 10,000 daltons (Millipore, Bedford, MA)according to the manufacturer's instructions.

For the Department of the polypeptide zalpha11CEE from free peptide by HER and any impurity suicidality proteins, combined concentrated fraction was subjected to chromatography on a column of 1.5×90 cm Sephadex S200 (Pharmacia, Piscataway, NJ), equilibrated and loaded in SFR at the speed of a current of 1.0 ml/min using a BioCad Sprint. Fractions of 1.5 ml were collected throughout chromatography and absorption at 280 and 215 nm were subjected to monitoring. The fraction of the peak elution characterized by electrophoresis in LTO-page with silver staining and were United only by the most pure fractions. This material was purified polypeptide zalpha11CEE.

This purified material was subjected finally clearing on which alance 4 ml ActiClean Etox (Sterogene) to remove any remaining endotoxin. The sample was passed through a balanced SFR column under the action of gravity or four times, then the column was washed once with 3 ml SPR and this wash was combined with the "treated" sample. Then this material was sterile filtered (0.2 μm) and stored at -80°to prepare aliquot.

On the Western blokirobvannii colored Kumasi blue and silver gels electrophoresis LTO-page polypeptide zalpha11CEE was one major band with an average molecular weight of approximately 50,000 daltons. The mobility of this band was the same for pampering and non gels.

The protein concentration of purified material was determined according to the analysis using BSA (Pierce, Rockford, IL) and protein was divided into aliquots and stored at -80°in accordance with the normal procedures of the authors of the invention. On IEF-gels (isoelectric focusing) protein was moved with PI less than 4.5. The concentration of the polypeptide zalpha11CEE was 1.0 mg/ml

To obtain anti-HER-Sepharose layer volume 100 ml protein G-Sepharose (Pharmacia, Piscataway, NJ) were washed 3 times with 100 ml SPR, containing 0,02% sodium azide using a filter element of 0.45 μm Nalgene 500 ml Gel was washed 6,0 volumes of 200 mm triethanolamine, pH of 8.2 (tea, Sigma, St. Louis, MO) was added to an equal volume of a solution of antibodies against ITS containing 900 mg of antibody. After incubation over night at 4#x000B0; With unbound antibody was removed by washing the resin with 5 volumes of 200 mm tea, as described above. Resin resuspendable in 2 volumes of tea, was transferred to a suitable container and then added dimethylpyrimidin-2hcl (Pierce, Rockford, IL), dissolved in tea, to a final concentration of 36 mg/ml G-Sepharose gel. The gel was rocked at room temperature for 45 minutes and the liquid was removed using a filter element as described above. Then nonspecific sites on the gel were blocked by incubation for 10 minutes at room temperature with 5 volumes of 20 mm ethanolamine 200 mm tea. Then the gel was washed in 5 volumes SFR, containing 0,02% sodium azide, and stored in this solution at 4°C.

C. Purification of the zalpha11 polypeptide CFLAG from KSS 570

Unless otherwise noted, all operations were carried out at 4°C. the Following procedure was used for purification of the zalpha11 polypeptide containing C-terminal FLAG label® (FLG) (Sigma-Aldrich Co.), Thirty liters of conditioned medium from the cell factory was concentrated to 1.7 liters using a spiral cartridge Amicon S10Y3 on ProFlux A30. The solution proteasome inhibitor was added to the concentrated 1.7 l air-conditioned environment of the cell factory from transfected cells KSS 570 (see example 9) to final concentrations of 2.5 mm ethylendiaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis, MO), 0,003 mm leupeptin (Boehringer Mannhim, Indianapolis, IN), 0.001 mm of pepstatin (Boehringer Mannheim) and 0.4 mm Pefabloc (Boehringer Mannheim). Samples were taken for analysis and the total volume was frozen at -80°before cleaning. The concentration of the target protein, air-conditioned environment of the cell factory was determined by electrophoresis in LTO-SDS page and Western blot analysis with antibodies against FLAG® (Kodak), conjugated with HRP (horseradish peroxidase). Column 125 ml anti-FLAG® M2-agarose affinity gel (SIGMA-Aldrich Co.) poured into a glass column, Waters AP-5, 5 cm × 10 cm Column was Packed flowing and balanced on a BioCad Sprint (PerSeptive BioSystems, Framingham, MA) phosphate buffered saline (SFR) pH 7.4. Concentrated conditioned medium of cell factories thawed, sterile filtered with a filter of 0.2 microns, the pH was brought to 7.4, and then applied to the column during the night with a flow rate of 1 ml/min. and the Column washed with 10 column volumes (KO) taputeranga phosphate salt solution (SFR, pH 7.4), then suirable with the flow of eluent through the tube 250 ml SFR (pH 6.0)containing 0.5 mg/ml FLAG® (Sigma-Aldrich Co.) peptide, when the speed of the current 5 ml/min Used FLAG®the peptide has the sequence DYKDDDDK (SEQ ID NO:37). The column washed with 10 column volumes (KO) SFR, then suirable 5 column volumes of 0.2 M glycine, pH 3.0. pH eluruumi glycine column was brought to 7.0 with 2 column volumes HSFR, then balanced in SFR (pH 7.4). Fractions of 5 ml were collected throughout elsinoe chromatography and absorption at 280 and 215 nm were subjected to monitoring; passing through the column, and wash pools well maintained and analyzed. The fraction of the peak elution FLAG®-polypeptide was analyzed for the target protein electrophoresis in LTO-page with silver staining and Western blotting with conjugated antibody anti-FLAG®-HRP. Interest fraction elution of the polypeptide were pooled and concentrated with 80 to 12 ml using a centrifugal concentrator with a membrane, cut-off molecular weight of 10,000 daltons (Millipore, Bedford, MA)according to the manufacturer's instructions.

For the Department zalpha11CFLG from other suicidality proteins, merged fraction elution of the polypeptide was subjected to purification on a POROS HQ-50 (strong anion exchange column from PerSeptive BioSystems, Framingham, MA) at pH 8.0. Column 1,0×6.0 cm was flooded and flowing Packed on a BioCad Sprint. The column was loaded by the counterion and then balanced in 20 mm Tris pH 8.0 (Tris(hydroxyethylaminomethyl)). The sample was diluted 1:13 (to reduce ionic strength SFR), then applied to the column POROS HQ-50 at the speed of a current of 5 ml/min. and the Column washed with 10 column volumes (KO) 20 mm Tris pH 8.0, was then suirable 40 column volume gradient of 20 mm Tris/1 M sodium chloride (NaCl) at a speed of DC 10 ml/min Fraction ,5 ml were collected throughout chromatography and absorption at 280 and 215 nm were subjected to monitoring, The fraction of the peak elution were analyzed by electrophoresis in LTO-page with silver staining. Interest fractions were combined and concentrated to 1.5-2 ml using a centrifugal concentrator with a membrane, cut-off molecular weight of 10,000 daltons (Millipore, Bedford, MA)according to the manufacturer's instructions.

For the Department of the polypeptide zalpha11CFLG from free FLAG peptide® and any impurity suicidality United proteins concentrated fraction was subjected to chromatography on a column of 1.5×90 cm Sephacryl S200 (Pharmacia, Piscataway, NJ), equilibrated and loaded in SFR at the speed of a current of 1.0 ml/min using a BioCad Sprint. Fractions of 1.5 ml were collected throughout chromatography and absorption at 280 and 215 nm were subjected to monitoring. The fraction of the peak elution characterized by electrophoresis in LTO-page with silver staining and were United only by the most pure fractions. This material was purified polypeptide zalpha11CFLG.

This purified material was subjected finally purification on a column of 4 ml ActiClean Etox (Sterogene) to remove any remaining endotoxin. The sample was passed through a balanced SFR column under the action of gravity or four times, then the column was washed once with 3 ml SPR and this wash was combined with the "treated" sample. Then this material was sterile filtered (0.2 μm) and stored at -80°to whom prigotovleniya of the aliquot.

On the Western blokirobvannii colored Kumasi blue and silver gels electrophoresis LTO-page polypeptide zalpha11CFLG was one major band with an average molecular weight of approximately 50,000 daltons. The mobility of this band was the same for pampering and non gels.

The protein concentration of purified material was determined according to the analysis using BSA (Pierce, Rockford, IL) and protein was divided into aliquots and stored at -80°in accordance with the normal procedures of the authors of the invention. On IEF-gels (isoelectric focusing) protein was moved with PI less than 4.5. The concentration of the polypeptide zalpha11CFLG was 1.2 mg/ml

C. Purification of the polypeptide zalpha11-Fc4 from transfected cells KSS 570

Unless otherwise noted, all operations were carried out at 4°C. the Following procedure was used for purification of the zalpha11 polypeptide containing C-terminal fusion with IgG/Fc man (zalpha11-Fc4; examples 8 and 9). 12000 ml of conditioned medium from cells KSS 570, transfected zalpha11-Fc4 (example 9), was filtered through a sterilizing filter of 0.2 mm and then was added a solution of protease inhibitors to a final concentration of 0.001 mm leupeptin (Boehringer Mannheim, Indianapolis, IN), 0.001 mm of pepstatin (Boehringer Mannheim) and 0.4 mm Pefabloc (Boehringer Mannheim). Protein G-Sepharose (layer volume 6 ml, Pharmacia Biotech) was Packed and washed with 500 ml SPR (Gibco/BRL). Supplemented conditioners the new environment was passed through the column at a speed of DC 10 ml/min followed by washing 1000 ml SPR (BRL/Gibco). Polypeptide zalpha11-Fc4 was suirable from the column with 0.1 M glycine pH 3.5 and fractions of 2 ml were collected directly in 0.2 ml of 2 M Tris pH 8.0 to bring the final pH to 7.0 in these fractions.

Erwerbende fraction characterized by electrophoresis in LTO-page and Western blotting with antibodies against Fc man (Amersham). Western blot analysis of reducing gels electrophoresis LTO-SDS page revealed immunoreactive protein of approximately 80000 kDa in fractions 2-10. Painted silver gel electrophoresis LTO-page also showed the polypeptide zalpha11-Fc4 80000 "Yes in fractions 2-10. Fractions 2-10 together.

The protein concentration of the combined fractions was determined according to the analysis using BSA (Pierce, Rockford, IL) and the material was divided into aliquots and stored at -80°in accordance with the usual procedures of applicants. The protein concentration of the combined fractions was 0.26 mg/ml

Example 11

Test using soluble zalpha11 receptor zalpha11CEE, soluble receptors zalpha11CFLG and zalpha11-Fc4 (mutant) in a competitive inhibitor analysis

Cells BaF3/-zalpha11 Unscrew and washed in not containing mlL-3 environment. The cells were turned off and washed 3 times to ensure removal of mlL-3. The cells are then considered in hemocytometer. Cells were planted in 96-well plate at 5000 cells per well in a volume of 100 μl per well using not containing mlL-3 environment.

As kondicionirovaniya environment from activated spleen cells monkeys, and CD3+ selected human cells, described in example 5 above, was added in separate experiments at concentrations 50%, 25%, 12,5%, 6,25%, 3,125%, 1,5%, 0,75% and the 0.375%, soluble zalpha11 receptor or without them (structures CEE, C-FLAG and Fc4; see example 9 and 10) at 10 µg/ml Total test was 200 ml

The test plates were incubated at 37°C, 5% CO2within 3 days, after which was added Alamar Blue (Accumed) at 20 μl per well. Tablets are again incubated at 37°C, 5% CO2within 24 hours. Tablets read on a tablet reader Fmax™ (Molecular Devices)as described in example 2. The results showed a complete inhibition of cell growth of each of the various structures of soluble zalpha11 receptor with 10 μg/ml, confirming that the factor in each sample was specific for the zalpha11 receptor.

Curves titration with dilutions of these soluble receptors were also determined using the above described test. As zalpha11CEE and zalpha11CFLG soluble zalpha11 receptor were able to completely inhibit the growth of such a low concentration as 20 ng/ml of soluble Mutant receptor zalpha11-Fc4 was effective only at 1.5 µg/ml

Example 12

Test secretory trap

Test secretory trap used to identify a cDNA for zalpha11 ligand. Positive DNA pools were obtained from the expression clone is written in example 7. Positive DNA pools 250 clones were transfusional cells KSS in the format of a 96-hole plansheta and air-conditioned environment used in the test of proliferation cell line BaF3/zalpha11 described in examples 4 and 5. Multiple DNA pools were positive activity, which was repeated and neutralized soluble zalpha11 receptor (see example 11).

One of the positive DNA pools, S, transfusional cells COS-7 12-hole format, using the method with Lipofectamine™described below. Then test the secretion trap was performed using soluble zalpha11 receptor (labeled on the C-end-labeled Glu-Glu with bioterrorism or without biotinidase; labeled on the C-end of the FLAG; or m RS-soluble zalpha11 receptor) (example 9) to test the direct link between potential zalpha11 ligand-receptor in the pool S and zalpha11 receptor (see below). The result was positive. Thus, DNA pool C was electroporative in E. coli and individual colonies were viscerale in ten 96-well plates. The tablets were shaken at 37°C for 24 hours and then minipreparation DNA (QiaPrep™ 96 Turbo Miniprep Kit; Qiagen) were prepared in 96-well format using TomTech Quadra 9600. Then plasmid DNA was combined in the format of rows and columns, transfusional in COS cells and then determined the positive pools when pamoxicillin traps as is described below.

Transfection of COS cells

Transfection of COS cells was performed as follows: Mix 3 ál of combined DNA and 5 μl of Lipofectamine™ 92 μl serum-free DMEM medium (55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg transferrin, 2.5 mg insulin, 1 μg selenium and 5 mg of fetuin in 500 ml DMEM), incubated at room temperature for 30 minutes and then add 400 ál Bassington medium DMEM. This mixture of 500 μl was added 1.5×105of COS cells per well planted at 12-hole tablet for tissue culture, and incubated for 5 hours at 37°C. 500 μl of 20% AppData% FCS-DMEM (100 ml FCS, 55 mg sodium pyruvate and 146 mg L-glutamine in 500 ml DMEM) was added and incubated overnight.

Test secretory trap

Test the secretion trap was performed as follows: the medium was washed from the cells using SPR and then fixed for 15 minutes with 1.8% formaldehyde in SFR. Then cells were washed in TNT (0.1 M Tris-HCl, 0.15 M NaCl and 0.05% tween-20 in H2O) and impregnated with 0.1% Triton-X in STR for 15 minutes and again washed with TNT. Cells were blocked for 1 hour using TNB (0.1 M Tris-HCl, 0.15 M NaCl and 0.5% blokiruyuschij reagent (NEN Renaissance TSA-Direct Kit) in H2O) and again washed with TNT. When using a biotinylated protein, the cells were blocked for 15-minute incubation with Avidya and then Biotin (Vector Labs), with washing between n the mi TNT. Depending on which soluble receptor was used, cells were incubated for 1 hour with: (A) 1-3 µg/ml fused protein of soluble zalpha11 zlh11-receptor-RS (example 10); () 3 µg/ml of soluble zalpha11 receptor labelled With the end FLAG, zalpha11CFLG (example 10); (C) 3 µg/ml of soluble zalpha11 receptor labeled on the end of the Glu-Glu, zalpha11CEE (example 10); or (D) 3 μg/ml biotinylated soluble zalpha11-receptor zalpha11CEE in TNB. Then cells were washed in TNT. Depending on which soluble receptor was used, cells were incubated for another hour with: (A) diluted 1:200 goat antibodies against human Ig-HRP (Fc specific); (C) diluted 1:1000 M2-HRP; (C) diluted 1:1000 antibody against Glu-Glu-HRP; or (D) diluted 1:300 complex streptavidin-HRP (set NEN) in TNB. Again cells were washed in TNT.

Positive binding was detected the fluorescein-termenum reagent, diluted 1:50 in buffer for cultivation (set NEN) and incubated for 4-6 minutes and washed with TNT. Cells were conserved environment for mount preparations Vectashield Mounting Media (Vector Labs, Burlingame, CA), diluted 1:5 in TNT. Cells were visualized using TCP filter on the fluorescent microscope.

Example 13

Chromosomal assignment and determine where in the chromosome to the gene zalpha11 ligand

The gene for zalpha11 ligand mapped on chromosome 4 is the use of commercially available version of the toolbar "Stanford G3 Radiation Hybrid Mapping Panel" (Research Genetics, Inc., Huntsville, AL). "Stanford G3 R.H. Panel contains DNA from each of 83 radiation (irradiated) hybrid clone a whole human genome, plus two control DNA (RM-donor and A3-recipient). Publicly available WWW server (http://shgc-www.stanford.edu) allows to determine the chromosomal localization of markers.

For mapping gene zalpha11 ligand with the panel "Stanford G3 RH Panel" reactions of 20 ál) were placed in 96-well microtiter tablet (Stratagene, La Jolla, CA) and used in thermocycler "RoboCycler Gradient 96" (Stratagene). Each of the 85 PCR reactions consisted of 2 μl of 10X KlenTaq PCR reaction buffer (Clontech Laboratories, Inc., Palo Alto, CA), and 1.6 μl of dNTP mixture (2.5 mm each, PERKIN-ELMER, Foster City, CA), 1 μl sense primer, ZC 22050 (SEQ ID NO:42), 1 μl antisense primer ZC22051 (SEQ ID NO:43), 2 μl "RediLoad" (Research Genetics, Inc., Huntsville, AL), and 0.4 μl of 50X mixture of Advantage KlenTaq Polymerase (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and ddH2O for a total volume of 20 µl. To the reaction was layered equal amount of mineral oil and they were sealed. Conditions for PCR cycler were as follows: 1 initial cycle of 4 min denaturation at 94°C, 35 cycles of 45 seconds denaturation at 94°C, 45 seconds of annealing at 60°and 1 minute and 15 seconds elongation at 72°C; with a final 1 cycle extension of 7 minutes at 72°C. the Reactions were separated by electrophoresis on 2% agarose gel (Life Technologies, Gaithersburg, MD).

Results pokazatelej gene zalpha11 ligand relative to the frame marker SHGC-12342 IL-2 with LOD-score > 12 at a distance of 6 cR 10000 (approximately 180 TPN) from the marker. The use of surrounding markers puts gene zalpha11 ligand in the 4q27 region on the integrated map LDB chromosome 4 (The Genetic Location Database, University of Southhampton, WWW server: http://cedar.geneties.soton.ac.uk/public html/.

Example 14

Identification and cloning of murine zalpha11 ligand

The use of EST sequences to obtain a full-sized mouse zalpha11 ligand

A. EST sequence murine zalpha11 ligand

Search in the database with the DNA sequence of the human zalpha11 ligand (SEQ ID NO:1) as request, the mouse EST (marker expressed sequence) (EST1483966) was identified as a potential partial sequence for murine zalpha11 ligand. EST1483966 is a mouse genomic fragment in which a peptide sequence derived from two potential exons, has shared high sequence identity with a peptide segment of the human zalpha11 ligand (amino acid 13 (lie) - amino acid 80 (Gin) SEQ ID NO:2).

C. PCR screening of mouse marathon-cDNA-panel

Eleven murine samples Marathon-cDNA (Clontech) podurgiel screening using PCR, as described below. Mouse sample marathon cDNA was obtained from the tissues of the brain, pancreas, kidney, placenta, salivary glands, skin, testis, uterus, embryo and spleen. Authors g is touili them in the laboratory with the use of the kit for the amplification Marathon™ -cDNA (Clontech) according to the manufacturer's instructions. Based on the EST sequences of the two PCR primers, ZC22056 (SEQ ID NO:44) and ZC22057 (SEQ ID NO:45) was used to identify the source of mouse zalpha11 ligand using PCR Conditions PCR reactions were as follows: 94°C for 2 minutes; 35 cycles at 94°C for 30 seconds, 68°C for 2 minutes; then 68°C for 4 minutes; then soaking in 10°C. PCR products were subjected to electrophoresis on 1% agarose gel. Strong band 150 BP, representing amplificatory "DNA fragment was visualized. This indicates that marathon-cDNA spleen is the source for cDNA cloning zalpha11 ligand. Mouse marathon-spleen cDNA contained positive "DNA, which is then identified by sequence analysis as a partial kankala murine zalpha11 ligand.

C. Composite sequence for full-length cDNA was generated using 5'- and 3'-RACE (rapid amplification of cDNA ends)

5' and 3' flanking sequences of a partial cDNA sequence of mouse zalpha11 ligand was obtained by amplification of the 5'- and 3'-RACE. Two rounds of PCR amplification with integrated primers were performed with additional gene-specific oligo-primers ZC22205 (SEQ ID NO:46) and ZC22206 (SEQ ID NO:47), ZC22056 (SEQ ID NO:44) and ZC22057 (SEQ ID NO:45) and two adaptorname oligo-what ramarama ZC9739 (SEQ ID NO:48) and ZC9719 (SEQ ID NO:49). PCR reactions were performed as follows: 94°C for 2 minutes; 35 cycles at 94°C for 30 seconds, 68°C for 2 minutes; then 68°C for 4 minutes; then soaking in 10°C. PCR products were subjected to electrophoresis on 1% agarose gel, and was identified by 5'-CASA-product of approximately 300 BP and 3'-RACE product of approximately 800 BP These fragments were isolated with the use of the kit for the extraction of the Qiaquick gel™ (Qiagen).

Purified PCR products are sequenced using the following primers: ZC9719 (SEQ ID NO:49), ZC22205 (SEQ ID NO:46) and ZC22206 (SEQ ID NO:47). Preliminary compositional full sequence of mouse zalpha11 ligand was identified by combining the 5'- and 3'-RACE fragments. Full-size mouse clone was isolated as described in example 15 below.

Example 15

The allocation of a cDNA clone of mouse zalpha11 library from activated murine spleen

A. Murine primary source used to distinguish murine zalpha11 ligand

Mouse spleen of BALB/c mice were collected and crushed between the object glass with frosted edges of obtaining a cell suspension. The output of the selected primary mouse cells was 6,4x108cells before selection, described below.

The spleen cells suspended in 9.6 ml of MACS buffer (STR, 0.5% EDTA, 2 mm EDTA). 1.6 ml of cell suspension shock the Yali was added 0.4 ml of CD90 (h1.2)-microgranules (Miltenyi Biotec). The mixture is incubated for 15 minutes at 4°C. These cells labeled granules CD90, washed with 30 ml of MACS buffer and then resuspendable in 2 ml of MACS buffer.

VS+ column (Miltenyi) were prepared in accordance with the manufacturer's instructions. Then VS+ column placed in a magnetic field VarioMACS™ (Miltenyi). The column was balanced with 5 ml of MACS buffer. Selected primary mouse cells was applied then this column. CD3-negative cells were made to pass through the column. The column is washed with 9 ml (3×3 ml MACS buffer. Then the column was removed from the magnet and placed in a falcon tube 15 ml CD90+ cells were suirable by adding 5 ml of MACS buffer to the column and the bound cells were washed using a piston (plunger)provided by the manufacturer. Incubation of these cells with CD90-magnetic granules, leaching and phase VS+ column (incubation-elution), described above, was repeated again. Received CD90+ fraction from column 2 divisions combined. The total yield of CD90+ selected cells of the mouse spleen was 1×108cells.

Sample United CD90+ selected mouse cells was removed for staining and sorting cell sorting device with fluorescent antibodies (FACS) to assess their purity. PE-conjugated antibody against mouse CD3ε hamster (PharMingen) were used for staining and sorting CD90+ selected cells. Mouse CD90+ selected to etki were 93% CD3+ cells, that suggests that these cells were 93% of T-cells.

Mouse CD90+ selected cells were activated by incubation 3×106cells/ml in RPMI + 5% FCS + PMA 10 ng/ml and Ionomycin 0.5 μg/ml (Calbiochem) overnight at 37°C. the Supernatant from these activated CD90+ selected mouse cells tested on the activity of zalpha11 ligand, as described below. In addition, activated CD90+ selected mouse cells were used to produce cDNA library as described in example 16 below.

Example 16

Cloning of mouse zalpha11 ligand from the library mouse CD90+ selected cells

Screening libraries of DNA activated primary mouse CD90+ selected cells revealed a dedicated DNA, which is a new member of the family of cytokines with the structure chetyrekhmernogo beam. This cDNA encodes the mouse ortholog of zalpha11 ligand person. This cDNA was identified by hybridization screening.

A. Vector for constructing CD90+ selected library

Vector pZP7N used to construct CD90+ selected library (see example 6A).

C. obtaining a cDNA library of activated primary mouse CD90+ selected cells

Approximately 1.5×108primary mouse CD90+ selected cells, stimulated in a mixture of ionomycin/PMA (example 15), were isolated by centrifugation. Total RNA was isolated by the C sediment cells and converted into double-stranded cDNA, as described in example 6B. Then this DNA was transfusional cells KSS, as described in example 6B, and proliferation was assessed using fluorescence analysis "Alamar blue" (example 2B).

For the purpose of screening the library by cloning with secretory trap was necessary comprehensive amplificatory form library for the transfection of cells COS-7. 4.8 million clones were sown at 110 cups 15 cm LB-agar supplemented with 100 µg/ml ampicillin, 10 μg/ml methicillin. After growing on plates over night at 37°bacteria were collected by scratching and besieged. Plasmid DNA was extracted from the besieged bacteria using Nucleobond-giga™ (Clontech) according to the manufacturer's instructions. Then, this plasmid was used for transfection of cells COS-7 on slides and subjected to screening using the method of secretory trap, described below (example 17).

C. Screening of activated murine cDNA library

Approximately 5×1010clones were sown on 10 LB/Amp Maxi-cups. Colonies were removed, was denaturiruet neutralized and were made using standard procedures (Sambrook, J. et al., supra). Fifty nanograms 5'RACE PCR fragment of 300 BP (example 14) were labeled32P using a set of tagging random primers to Prime-ltr RmT (Stratagene). 10 filters hybridized with this labeled probe at 65° With during the night using the solution for ExpressHyb hybridization™ (Clontech). Then the filters were washed sequentially at 60°C for 1 hour three times with 0.1×SSC (30 mm NaCl, 3 mm sodium citrate, pH 7.0), 0.1% of LTOs; and then at 65°C for 1 hour. Filters were exposed at -80°during the night and the x-ray film showed. Agar plugs containing positive colonies were removed and these clones were planted on LB/Amp-Cup 10 see Then colonies were removed with the filter and again hybridized according to the procedure described above.

One DNA clone, named M11L/pZP7, was isolated and sequenced using the following primers: ZC14063 (SEQ ID NO:50), ZC5020 (SEQ ID NO:51), ZC22421 (SEQ ID NO:52), ZC22604 (SEQ ID NO:53) and ZC22641 (SEQ ID NO:54), the Polynucleotide sequence of this clone is a full-sized mouse zalpha11-ligand (SEQ ID NO:55) and is consistent with the composite sequence derived from the 5'- and 3'-RACE products. The corresponding amino acid sequence for murine zalpha11 ligand shown in SEQ ID NO:56.

Example 17

Murine zalpha11 ligand binds to soluble zalpha11 receptor human test secretory trap

DNA for mouse clone M11L/pZP7 was transfusional in COS cells and the binding of soluble zalpha11 receptor human fused protein zalpha11-Fc4 (example 10C) with transfitsirovannykh COS cells was tested using the test secretor the second trap (example 12). This test confirmed that murine zalpha11 ligand binds to soluble zalpha11 receptor human.

Transfection of COS cells was performed as in example 12, with the use of 0.7 µg DNA 11L/pZP7 (example 16) in 3 μl.

Test the secretion trap was performed as in example 12 using 1 µg/ml fused protein of soluble zalpha11 receptor human RS (example 10C) in TNB and 1:200 diluted goat antibodies against human Ig-HRP (Fc specific) in TNB for the detected antibodies. Positive binding of soluble zalpha11 receptor person with a made-fixed cells were detected the fluorescein-termenum reagent as in example 12. Cells were conserved and visualized according to example 12.

A positive result was indicated that murine zalpha11 ligand binds to soluble zalpha11 receptor human.

Example 18

Expression of murine zalpha11 ligand in mammalian cells

A. Designing expressing vector M11L/pZP9

Prepared expressing the vector for the expression of mouse zalpha11 ligand in mammalian cells. Generated PCR DNA fragment zalpha11 ligand 500 BP was created using ZC22283 (SEQ ID NO:57) and ZC22284 (SEQ ID NO:58) as PCR primers to amplify the coding region of mouse zalpha11 ligand and site enzyme Xhol and Xbal. Clone M11L/pZP7 mouse zalpha11 ligand (example 16) is the objects of study were as matrix. Conditions for PCR reactions were as follows: 94°C for 2 minutes; 25 cycles at 94°C for 30 seconds, 68°C for 2 minutes; then 68°C for 4 minutes; then soaking in 10°C. a Band of the predicted size of approximately 500 BP were visualized by gel-electrophoresis on 1% agarose, cut out and DNA was purified using a purification system Qiaexll™ (Qiagen) in accordance with the manufacturer's instructions. Purified DNA was digested Xhol and Xbal (Boehringer Mannheim) at 37°C for 2 hours. Then this DNA was isolated from the gel and purified according to the above procedure.

The cut DNA was subcloned into plasmid pZP9, which was cut Xhol and Xbal (Boehringer Mannheim). Plasmid pZP9 is expressing vector mammals containing expression cassette having a promoter mouse metallothionein-1 (MT-1), multiple restriction sites for embedding the coding sequence and terminator of human growth hormone. This plasmid is also the beginning of replication of E. coli, the expression unit selective marker expression mammals, with the promoter, enhancer and early SV40 replication, a DHFR gene, and the SV40 terminator.

Approximately 30 ng split restrictase fragment of mouse zalpha11 ligand and about 10 ng of the split vector pZP9 ligated at room temperature for 2 hours. Two μg of the reaction Legerova the Oia transformed into INVaF'competent cells (Invitrogen) according to the manufacturer's Protocol and were sown on LB-cups, containing 50 μg/ml ampicillin, and incubated at 37°With during the night. Colonies were subjected to screening restriction fragments length polymorphism analysis using Xhol and Xbal (Boehringer Mannheim) DNA obtained from liquid cultures of individual colonies. The sequencing analysis confirmed that the sequence of the inserts of positive clones is a sequence of murine zalpha11 ligand. Large-scale obtaining plasmids was performed using the kit Qiagen® Maxi prep kit (Qiagen) according to the manufacturer's instructions. Expressing the vector, which contains the murine zalpha11 ligand, was named M11L/pZP9.

C. Expression of murine zalpha11 ligand in mammals

Cells KSS 570 (ATSS No. CRL-10314) were sown in cups for tissue culture 10 cm and gave them to grow to approximately 20% of confluently over night at 37°C, 5% CO2in the DMEM/FCS (DMEM, Gibco/BRL High glucose media; Gibco BRL, Gaithersburg, MD), 5% fetal serum (Hyclone, Logan, UT), 1 mm L-glutamine (JRH Biosciences, Lenexa, KS), 1 mm sodium pyruvate (Gibco BRL)). Then the cells were transfusional the plasmid M11L/pZP9 (example 18A) using the set of stable Saro4-transfection mammals (Stratagene) according to the manufacturer's instructions.

Later, one day after transfection the cells were split 1:10 and 1:20 in selective medium (DMEM/FCS with the addition of 1 μm methotrexate (Sigma Chemical Co., St. Louis, MO)) in the cups 150 mm Environment on this is x cells was replaced with fresh selective medium at day 5 after transfection. After approximately 10 days after transfection resistant to methotrexate colonies were trypsinization and cells were combined and were sown in the culture flask for large-scale retrieval. As soon as these cells multiply to approximately 90% of confluently, washed SFR three times and cultured with serum-free medium ESTEP2 (DMEM (Gibco BRL), 0.11 g/l sodium pyruvate, 3.7 g/l NaHCO3, 2.5 mg/l insulin, 5 mg/l transferrin, pH 7.0) to obtain the air-conditioned environment. Conditioned medium was collected three days later and was placed in the test proliferation of BaF3 using Alamar Blue as described in example 19 below.

Example 19

Murine zalpha11 ligand activates zalpha11 receptor of human rights in BaFS-test using Alamar Blue

Cell proliferation BaF3/zalpha11 (example 4 and 5B) was estimated using serum-free conditioned medium from KSS-cells expressing mouse zalpha11 ligand (example 18).

Cells BaF3/zalpha11 was turned off, washed and were sown in not containing mlL-3 environment, as described in example 5B.

Cell proliferation BaF3/zalpha11 was estimated using serum-free conditioned medium from cells KSS expressing mouse zalpha11 ligand (example 18). Air-conditioned environment bred not containing mlL-3 medium to concentrations: 50%, 25%, 12,5%, 6,25%, 3,125%, 1,5%, 0,75% and the 0.375%. Analysis of cell proliferation was performed as in example 5, the.

The results confirmed the proliferative response of cells BaF3/zalpha11 on murine zalpha11 ligand. This reaction, as measured, was approximately 5 times higher than the background at a concentration of 50%.

Example 20

Zalpha11 ligand activates zalphall-receptor of human rights in luciferase test

A. Construction of cell line BaF3/KZ134/zalpha11

The KZ134 plasmid was designed with complementary oligonucleotides ZC12749 (SEQ ID NO:59) and ZC12748 (SEQ ID NO:60), which contain elements of binding of the transcription factor STAT of 4 genes: modified induced element of c-fos Sis (m67SIE or hSIE) (Sadowski, H. et al., Science 261:1739-1744, 1993), the p21 SIE1 from the P21 gene WAFI (Chin, Y. et al., Science 272:719-722, 1996), the sensing element gene β-casein breast cancer (Schmitt-Ney, M. et al., Mol. Cell. Biol. 11:3745-3755, 1991) and inducible element STAT gene Fcg Rl (Seidel, H. et al., Proc. Natl. Acad. Sd. 92:3041-3045, 1995). These oligonucleotides contain Asp718-Xhol-compatible ends and ligated using standard methods in the recipient reporter vector Firefly c-fos-promoter (now, L.K. et al., J. Biol. Chem. 273:6229-6232, 1998), cleaved with the same enzymes and containing breeding marker neomycin. The KZ134 plasmid was used for stable transfection of BaF3 cells using standard methods of transfection and selection with obtaining cell line BaF3/KZ134.

Stable indicator cell line BaF3/KZ134 expressing full-zalpa11-receptor, designed as in example 2A, using about 30 μg expressing vector zalpha11 described in example 3. Clones were diluted, were sown and were taken using standard methods. The clones were subjected to screening using a luciferase test (see example 20, below) using an air-conditioned environment zalpha11 ligand person as an inductor. Selected clones with the highest luciferase response (via luciferase STAT) and the lowest background. He was selected line of stable transfectants. This cell line was called BaF3/KZ134/zalpha11.

C. Human and mouse zalpha11-ligands activate zalpha11 receptor in luciferase test BaF3/KZ134/zalpha11

Cells BaF3/KZ134/zalpha11 Unscrew and washed in not containing mlL-3 environment. The cells were turned off and washed 3 times to ensure removal of mlL-3. The cells are then considered in hemocytometer. Cells were planted in 96-well format with approximately 30,000 cells per well in a volume of 100 μl per well using not containing mlL-3 environment. The same procedure was used for nitrostilbene cells BaF3/KZ134 for use as a control in subsequent analysis.

STAT-activation of cells BaF3/KZ134/zalpha11 was assessed using conditioned medium from (1) cells VNC, transfected zalpha11-ligand person (example 7), or (2) cells VNC, transfected murine zalpa11-ligand (example 18), or (4) not containing mlL-3 environment to measure the response of the control-only environment. The conditioned medium was diluted not containing mlL-3 medium RPMI to concentrations 50%, 25%, 12,5%, 6,25%, 3,125%, 1,5%, 0,75% and the 0.375%. 100 μl of the diluted conditioned medium was added to cells BaF3/KZ134/zalpha11. Analysis using conditioned medium was performed in parallel on the cells BaF3/KZ134 as a control. The total volume of the reaction mixture was 200 ál. The test cups were incubated at 37°C, 5% CO2within 24 hours, after which these cells were besieged by centrifugation at 2000 rpm for 10 minutes and the medium was aspirated and added 25 μl lisanova buffer (Promega). After 10 minutes at room temperature, the Cup was measured on the activation of STAT-reporter construct by reading them on luminometer (Labsystems Luminoskan, model RS), which was added 40 μl of luciferase substrate test (Promega) with five seconds of integration.

The results confirmed STAT-reporter response of cells BaF3/KZ134/zalpha11 on human zalpha11 ligand. This response, as measured, was approximately 50-fold in comparison with control only-medium at a concentration of 50%. STAT activation in response to zalpha11 ligand man was absent in untreated control cells BaF3/KZ134 that indicates that this response is mediated zalpha11 receptor.

The results also confirmed the STAT-reporter response the cells BaF3/KZ134/zalpha11 on murine zalpha11 ligand. This response, as measured, was approximately 40-fold over the control environment at a concentration of 50%. In addition, STAT activation in response to murine zalpha11 ligand was evident (approximately 5-fold) on nitrostilbene control cells BaF3/KZ134, suggesting that murine BaF3 cells may be endogenous murine receptor.

Example 21

Murine zalpha11 ligand is active in the analysis of mouse bone marrow

A. Allocation of unattached cells in the bone marrow low density

Fresh aspirate mouse femur (bone marrow) were obtained from 6-10-week-old male mice of BALB/C or C57BL/6. Then the bone marrow were washed in RPMI+10% FCS (JRH, Lenexa KS; Hyclone, Logan UT) and suspended in RPMI+10% FCS in suspension of whole bone marrow cells. Then the suspension of whole bone marrow cells were subjected to density gradient (Nycoprep, 1.077, Animal; Gibco BRL) for enrichment in relation to having a low density mainly of mononuclear cells in the following way: a suspension of whole bone marrow cells (about 8 ml) was carefully pietravalle on the upper surface of approximately 5 ml Nycoprep gradient solutions in a conical tube 15 ml and then centrifuged at 600xg for 20 minutes. Then the interfacial layer containing mononuclear cell low-density, removed, washed with excess of RPMI+10% FCS and besieged zentrifugenbau is receiving at 400x g for 5-10 minutes. This sediment resuspendable in RPMI+10% FCS and were sown in the flask T-75 at approximately 106cells/ml and incubated at 37°C, 5% CO2for about 2 hours. The obtained cells in suspension were unattached cells in the bone marrow of low density (NA LD).

C. 96-well test

Cells mouse bone marrow NA LD were sown when 25000-45000 cells per well in 96-well tablets for tissue culture in RPMI+10% FCS + 1 ng/ml of factor murine stem cells (mSCF) (R&D Systems, Minneapolis, MN) plus 5% conditioned medium from one of the following cells: (1) cells KSS 570 expressing mouse zalpha11 ligand (example 18), (2) cells KSS 570 expressing human zalpha11 ligand (example 7), or (3) control cells KSS 570 containing the vector and not expressing any ligand. These cells were then subjected to various treatments with cytokines for the analysis of multiplication or differentiation of hematopoietic cells from the bone marrow. For this test sown murine bone marrow cells NA LD subjected to the action of IL-15 person (hlL-15) (R&D Systems) or one of the other panel of cytokines (R&D Systems). Serial dilution hlL-15 or other cytokines tested, with 2-fold serial dilution of the concentration of approximately 50 ng/ml to a concentration of approximately 6025 ng/ml After 8-12 days 96-well tests were evaluated at the points n the cell proliferation using a test with the dye Alamar blue, as described in example 5B.

C. the Results of the 96-well test in mouse bone marrow cells NA LD

Conditioned medium from cells KSS expressing both murine and human zalpha11 ligand, acted synergistically with hlL-15 in the promotion of the breeding population of hematopoietic cells in NA LD-cells mouse bone marrow. This is a reproduction of hematopoietic cells was not detected air-conditioned control cells KSS medium plus IL-15. The population of hematopoietic cells, multiplied mouse zalphall-ligand with hlL-15, and the population of hematopoietic cells, multiplied human zalpha11-ligand with hlL-15, additionally were propagated in cell culture. These hematopoietic cells were stained with phycoerythrin-labeled antibody against Pan NK-cells (Pharmingen) and were subjected to analysis of flow cytometry, which showed that the multiplied cells stained positive for this marker of natural killer cells (NK-cells).

The same 96-well test was performed using fresh cells of human bone marrow obtained from Poietic Technologies, Gaithersburg, MD. Again, in combination with IL-15, mouse and human zalpha11 ligand multiplied the population of hematopoietic cells, which stained positive for the marker of NK-cells using the above-described antibodies.

Example 22

Design to obtain with the holding zalpha11 ligand person transgenic mice

A. Design for the expression of zalpha11 ligand man from the liver-specific promoter (MT-1

Were designed oligonucleotides to generate a PCR fragment containing a consensus Kozak sequence and coding region zalpha11 ligand person. These oligonucleotides were designed with Fsel site at the 5'-end and a website Asel at the 3'end to facilitate cloning in (a) RMT-8, standard transgenic vector of the authors, or (b) pKF051, lymphoid-specific transgenic vector (example 22B).

PCR reactions were performed with 200 ng zalpha11 ligand person as a matrix (example 7) and oligonucleotides ZC22143 (SEQ ID NO:61) and ZC22144 (SEQ ID NO:62). Conditions for PCR reactions were as follows: 95°C for 5 minutes, was added cDNA polymerase Advantage™ (Clontech); 15 cycles of 95°C for 60 seconds, 60°C for 60 seconds and 72°C for 90 seconds; and 72°C for 7 minutes. PCR products were separated by agarose gel electrophoresis and purified using a kit for the extraction of QiaQuick gel™ (Qiagen). The selected DNA fragment 488 BP were digested Fsel and Asel (Boehringer Mannheim), precipitated with ethanol and ligated in RMT-8, pre-split Fsel and Asel. Plasmid RMT-8, designed for expression of interest gene in the liver and other tissues of transgenic mice, contains an expression cassette, flanked 10 TPN 5'-DNA M is -1 and 7 TPN 3'-DNA (MT-1. This expression cassette contains a promoter (MT-1, insulin intron II of mouse, polylinker to embed the desired clone, and a poly-a sequence of human growth hormone (hGH).

About one microliter of each ligation reaction was electroporative in electrocompetent relatively DH10B ElectroMAX™ cells (GIBCO BRL, Gaithesburg, MD) according to the manufacturer's instructions and were sown on LB-cups, soderjashie 100 μg/ml ampicillin, and incubated overnight. Colonies were viscerale and were grown in LB-medium containing 100 μg/ml ampicillin. Minireport DNA has been extracted from clones and subjected to screening for insert zalpha11 ligand person restriction cleavage only EcoRl or a combination of the Fsel and Asel and then gel electrophoresis. Received makeprimary exact pMT-zalpha11 ligand person. Was obtained Sall fragment containing the 5'- and 3'-flanking sequences, the MT promoter-1, insulin intron II of the rat cDNA zalpha11 ligand man and a poly-a sequence of hGH to apply for microinjection into fertilized murine oocytes. Microinjection and obtaining transgenic mice was performed as described in Hogan, B. et al., Manipulating the Mouse Embryo. 2nded., Cold Spring Harbor Laboratory Press, NY, 1994.

Century Design for the expression of zalpha11 ligand man from lymphoid-specific promoter EμLCK

Designed oligonucleotide is to generate a PCR fragment, containing a consensus Kozak sequence and coding region zalpha11 ligand person. These oligonucleotides were designed with Fsel site at the 5'-end and a website Asel at the 3'end to facilitate cloning in pKF051, lymphoid-specific transgenic vector.

PCR reactions were performed with 200 ng zalpha11 ligand person as a matrix (example 7) and oligonucleotides ZC22143 (SEQ ID NO:61) and ZC22144 (SEQ ID NO:62). PCR reactions were performed using cDNA polymerase Advantage ™ (Clontech) under the following conditions: 95°C for 5 minutes, 15 cycles of 95°C for 60 seconds, 60°C for 60 seconds and 72°C for 90 seconds; and 72°C for 7 minutes. PCR products were purified as described above. The selected DNA fragment 488 BP were digested Fsel and Asel (Boehringer Mannheim), was besieged by ztenolol and ligated in pKF051, pre-split Fsel and Asel. Transgenic vector pKF051 obtained from RH (Iritani, B.M., et al., EMBO J. 16:7019-31, 1997) and contains the T-klecko-specific proximal promoter Ick a/T-specific enhancer of the heavy chain immunoglobulin μ, polylinker to embed the desired clone, and a mutated hGH gene that encodes the protein inactive growth factor (providing the 3'introns and polyadenylation signal).

About one microliter of each ligation reaction was electroporative, were sown, the clones were viscerale and were subjected to screening for insert zapha11 ligand person restriction cleavage, as explained above. Exact clone pKF051-zalpha11 ligand was confirmed by sequencing and were getting makeprimary this clone. Notl fragment containing the proximal Ick promoter and enhancer of the immunoglobulin μ EμLCK), cDNA zalpha11 ligand and the mutated hGH gene was obtained for use for microinjection into fertilized murine oocytes.

Example 23

Tissue distribution of mouse zalpha11 ligand

Northern blots of multiple murine tissues (Mouse, Mouse Embrio, Clontech; MB1010, MB1012 Origene) was probed to determine the tissue distribution of expression of murine zalpha11 ligand. PCR-derived probe approximately 484 BP amplified using plasmid M11L/pZP7 (example 16) as template and oligonucleotides ZC22283 (SEQ ID NO:57) and ZC22284 (SEQ ID NO:58) as primers. PCR amplification was carried out as follows: 1 cycle at 94°C for 1.0 min; 35 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 30 seconds; then 1 cycle at 72°C for 10 minutes. PCR products were visualized by electrophoresis on agarose gel and the PCR product approximately 484 BP was purified using a kit for the extraction of gels (Qiagen) according to the manufacturer's instructions. The probe was radioactively labeled using the kit for labeling REDIPRIME™ (Amersham) according to the manufacturer's instructions. Probe ocialis using NUCTRAP column™ push Column (Stratagene). The EXPRESSHYB solution™ (Clontech) was used to prehybridization and as a solution for hybridization to Northern blots. Hybridization took place overnight at 65°With application of the 106pulse/min/ml of labeled probe. Then the blots were washed three times in 2x SSC and 0.1% LTOs at room temperature, followed by 2 washes in 0,1x SSC and 0.1% LTOs at 55°C. Two transcripts of approximately 1.2 and 3.5 TPN observed in the testis. Higher running transcript was observed only in the thymus.

Master Dot Blot of mouse RNA (Clontech), which contained RNA from various tissues, which have been normalized with respect to the 8 genes "household"also probed and hybridized as described above. Expression was observed in the testis.

Example 24

Purification of unlabeled human and mouse zalpha11 ligand from KSS 570

Unless otherwise noted, all operations were carried out at 4°C. the Following procedure was used for the purification of human and mouse zalpha11 ligand from the conditioned media from cells KSS 570, transfected with a construct expressing either zalpha11 ligand person (example 25)or murine zalpha11 ligand (M11L/pZP9) (example 18). Conditioned medium was concentrated by standard methods. Concentrated conditioned medium (KM) sterile filtered through filters of 0.45 and 0.22 micron. Then this CPE is from diluted to a low ionic strength (≤ 2 mS) in 0.01 M HEPES (JRH Bioscience, Lenexa, KS) at pH 7.0. Then diluted to a low ionic strength KM was applied on the column 10×66 mm (6 ml) Poros HS 50 (PerSeptive BioSystems, Framingham, MA) overnight at 4 ml/min using a BioCAD SPRINT (Perceptive BioSystems). This column was washed with 10-20 column volumes (KO) 0.01 M HEPES pH 7.0. Then associated proteins speed was suirable 1 M NaCl (Mallinckrodt, Paris, KY) in 0.01 M HEPES pH 7.0 at 5 ml/min; fraction two ml were collected throughout chromatography and absorption at 280 and 215 nm were subjected to monitoring. Fraction peaks were analyzed using bioanalysis and using gel electrophoresis in SDS page-ordinator with silver staining (Geno Technology, St. Louis, MO) and Kumasi (Signna, St. Louis, MO). Fraction peaks were pooled, sterile filtered and diluted to ≤19 mS sulfate buffered saline (PBS, Gibco BRL), pH 7,2.

Then the diluted sample was applied at 2 ml/min using a BioCad SPRINT on the column, 0.8 ml Poros AL, which had either a soluble receptor zalpha11CFLAG (example 10B), or fused soluble receptor zalpha11-Fc4 (example 10C)immobilized on the resin (see below). Then the column was washed with at least 20 TO SFR at 10 ml/min and Then the column was quickly suirable injection 600 μl of 0.1 M glycine (Aminoacetic Acid; Glycocol, Spectrum, Gardena, CA) pH of 2.5 at the speed of a current of 10 ml/min with SFR on a BioCAD 700E. Fractions of 1 ml were collected for 6 seconds each, and immediately neutralized pH 55 μl of 2 M Tris (Trihydroxysilyl)aminomethane, EM Science, Gibbstown, NJ) pH of 8.8. The absorption at 280 and 215 nm were monitored throughout the chromatography.

Fraction peaks were analyzed using bioanalysis and electrophoresis in SDS page-ordinator with silver staining (Geno Technology), Kumasi (Sigma). Two bands of approximately 24 KD and 18 KD. watched on the painted silver, and colored Kumasi gels for murine zalpha11 ligand. A single band at approximately 18 KD can be seen on painted as silver and Kumasi gels for zalpha11 ligand person.

Immobilization of polypeptides soluble zalpha11 receptor human environment POROS AL

Prepared column Poros AL having immobilized soluble receptor zalpha11CFLAG (example 10B) or fused soluble receptor zalpha11-Fc4. Used approximately 3 mg of soluble receptor zalpha11CFLAG and approximately 10 mg of the slit receptor zalpha11-Fc4. All surgeries were performed at room temperature on a BioCAD 700E. Column 4,5×50 mm with the environment POROS AL Packed flowing at 2 M NaCl in accordance with the manufacturer's instructions. Then the column was balanced in a mixture of 1.1 M Na2SO4/500 mm Na-phosphate pH of 7.2. The receptor was concentrated to 4 mg/ml using a centrifugal concentrator Millipore 30 MWKO, then diluted 1:1 in a mixture of 1.1 M Na2SO4/50 mm Na-phosphate pH of 7.2. Through the column was passed at 2 ml/min a mixture of 1.1 M Na2SO42SO4/50 mm Na-phosphate pH 7.2 to 550 mm Na2SO4/50 mm Na-phosphate pH 7.2 for 5 mg/ml cyanoborohydride sodium. Then the column was kept for about 2 hours to complete immobilization. Then the column was balanced in 0.2 M TRIS pH 7.2 for 5 mg/ml cyanoborohydride sodium and allowed to stand for approximately 1 hour for closing the column, and Finally the column was balanced in SFR/0,02% sodium azide and stored at 4°s to use. Before using pre-column was suirable 0.1 M glycine for guarantees that were removed nonspecific proteins and that this column was not Vasilache immobilized soluble zalpha11 receptor human.

Example 25

Expression of zalpha11 ligand person in mammalian cells

A. Designing expressing vector PZMP11/zalpha11Lig

The expression plasmid containing the entire polynucleotide or part of polynucleotide encoding zalpha11 ligand man, designed by homologous recombination. A fragment of DNA zalpha11 ligand human (SEQ ID NO:63) was isolated using PCR. Two primers were used to obtain this fragment zalpha11 ligand person in a PCR reaction: (1) primer ZC22052 (SEQ ID NO:64), containing 40 BP of flanking sequence is eljnosti vector and 17 BP, the corresponding amino-end of the zalpha11 ligand person, and (2) primer ZC22053 (SEQ ID NO:65), containing 40 BP of the 3'-end corresponding to the flanking sequences of the vector, and 17 BP corresponding to carboxyl-end of the zalpha11 ligand person. Conditions for PCR reactions were as follows: 1 cycle at 94°C for 2.0 minutes; 25 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds; then 1 cycle at 72°C for 5 minutes, soaking 4°C. Ten μl of the 100 μl PCR reaction was subjected to electrophoresis on 0.8% LMP-agarose gel (Seplaque GTG) 1 x TBE-buffer for analysis, and observed the expected fragment of approximately 560 BP Remaining 90 μl of PCR reaction was besieged by adding 5 ál of 1 M NaCl and 250 μl of absolute ethanol to be used for recombination in the recipient vector pZMPH, as described below. The recipient plasmid pZMPH pre-cut Smal.

Plasmid pZMPH was obtained from the plasmid pCZR199 (described here, for example, in example 8). Plasmid pCZR199 is expressing vector mammals, containing the expression cassette with immediately early promoter of CMV, the consensus intron of variable regions of the heavy chain locus of the mouse immunoglobulin, multiple restriction sites for installation of coding sequences, a stop codon and a terminator of human growth hormone. This plasmas is Yes is also the beginning of replication E. coli, the expression unit of breeding marker mammals, with the promoter, enhancer and early SV40 replication, a DHFR gene, and the SV40 terminator. Vector pZMPH designed from pCZR199 and it includes replacement of the promoter of metallothionein immediate early CMV promoter and a Kozak sequence at the 5'end of the open reading frame.

One hundred microlitres competent yeast cells (S. cerevisiae) were combined with 10 μl of a mixture containing approximately 1 μg inserts zalpha11 ligand person, and 100 ng of Smal split vector pZMPH, and transferred into a cuvette for electroporation see 0,2 Mix the yeast/DNA was subjected to electrical pulses at 0.75 kV (5 kV/cm), an indeterminate number of ohms, 25 μf. To each cuvette was added 600 μl of 1.2 M sorbitol and the yeast sown in two aliquot of 300 μl on two cups of URA-D and incubated at 30°C.

After approximately 48 hours, the Ura+ yeast transformants from a single Cup resuspendable in 1 ml of N2On and off quickly for the deposition of yeast cells. Cellular precipitate resuspendable in 1 ml lisanova buffer (2% Triton X-100, 1% LTOs, 100 mm NaCl, 10 mm Tris, pH 8.0, 1 mm EDTA). 500 ál lizinoj mixture was added to the Eppendorf tube containing 300 μl acid washed glass granules and 200 ál of a mixture of phenol/chloroform was shaken on a vortex two or three times at intervals of 1 min, followed by 5-minute loosening in General is trifuge Eppendorf at maximum speed. Three hundred microlitres the aqueous phase was transferred into a fresh tube and DNA was besieged by 600 ál of ethanol (EtOH), followed by centrifugation for 10 minutes at 4°C. the precipitated DNA resuspendable in 100 μl of N2O.

Transformation electrocompetent the cells of E. coli (DH10B, Gibco BRL) was performed with 0.5-2 ml yeast DNA and 40 μl of DH10B cells. Cells were subjected to electrical pulses at 2.0 kV, 25 MF and 400 Ohms. After electroporation, 1 ml SOC (2% Bacto™ tripton (Difco, Detroit, Ml), 0.5% of yeast extract (Difco), 10 mm NaCl, 2.5 mm KCl, 10 mm MgCl2, 10 mm MgSO4, 20 mm glucose) were sown in the aliquot 250 ál four LB AMR Cup (LB-broth (Lennox), 1.8% Baktagir (Difco), 100 mg/l ampicillin).

Individual clones carrying the correct expression construct for zalpha11 ligand man, identified restriction cleavage to confirm the presence of inserts and to confirm that the various DNA sequences have been correctly connected to each other. Insert positive clones were subjected to sequence analysis. When obtaining large-scale plasmid DNA was isolated using a kit QIAGEN Maxi kit (Qiagen) according to the manufacturer's instructions.

C. Expression of zalpha11 ligand person in mammals

Cells KSS 570 (ATSS No. CRL-10314) were sown in Cup 10 cm tissue culture and gave them to grow to approximately 50-70% of confluently in those who tell night at 37° C, 5% CO2in the DMEM/FCS (DMEM, Gibco/BRL High Glucose, (GIBCO BRL, Gaithersburg, MD), 5% fetal calf serum (Hyclone, Logan, UT), 1 mm L-glutamine (JRH Biosciences, Lenexa, KS), 1 mm sodium pyruvate (Gibco BRL). Then the cells were transfusional the plasmid PZMPH/zalpha11Lig (example 25A) using Lipofectamine™ (Gibco BRL), in serum free (BS) medium (DMEM, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml of fetuin, 1% L-glutamine and 1% sodium pyruvate). Plasmid zalpha11-Fc4/pZMP6 (example 8B) were bred in test tubes 15 ml to a total final volume of 640 μl BS environment. 35 ml of Lipofectamine™ (Gibco BRL) was mixed with 605 μl of BS. This blend of Lipofectamine™ was added to the mixture of DNA and allowed to incubated for approximately 30 minutes at room temperature. Five milliliters BS-medium was added to the mixture of DNA: Lipofectamine™. Cells were washed once with 5 ml of BS-environment, was aspirated and added to the mixture of DNA: Lipofectamine™. Cells were incubated at 37°C for five hours, then in each Cup was added to 6.4 ml of medium DMEM/10% FCS, 1% PSN. The cups were incubated at 37°during the night and the mixture of DNA: Lipofectamine™ was replaced with fresh medium with 5% FCS/DMEM for the next day. At day 5 after transfection the cells were destroyed in the flask T-162 in selective medium (DMEM/5% FCS, 1% L-GLU, 1% Na-Pyr). Approximately 10 days after transfection two cultural cups 150 mm resistant to methotrexate colonies from each of the transfection was trypsinization and cells were combined and were sown in the flask T-162 and carried in large-scale culture. Air-conditioned environment of the large-scale culture was used for purification of the polypeptide zalpha11 ligand person as described in example 24.

Example 26

The design for containing murine zalpha11 ligand transgenic mice

A. construction for the expression of mouse zalpha11 ligand from lymphoid-specific promoter EμLCK

Were designed oligonucleotides to generate a PCR fragment containing a consensus Kozak sequence and coding region zalpha11 ligand mouse. These oligonucleotides were designed with Fsel site at the 5'-end and a website Asel at the 3'end to facilitate cloning in (a) pKFO51, lymphoid-specific transgenic vector, or (b) pTG12-8, standard transgenic vector of the authors.

PCR reactions were performed with 200 ng zalpha11 ligand mouse as a matrix (SEQ ID NO:55; example 16) and oligonucleotides ZC23115 (SEQ ID NO:66) and ZC23116 (SEQ ID NO:67). PCR reaction was performed using cDNA polymerase Advantage™ (Clontech) PCR conditions described in example 22B. The PCR product was isolated as described in example 22B. The selected DNA fragment of 440 BP were digested and ligated in pKF51, pre-split Fsel and Asel, as described in example 22 Century

About one microliter of each ligation reaction was electroporative, were sown, the clones were viscerale and were subjected to screening for insert zalpha11 ligand person, as the description is about in example 22. The correct clone FO51-zlh11-ligand was confirmed by sequencing and got maxiprep this clone. Notl fragment containing the proximal promoter Ick, enhancer of immunoglobulin μ, cDNA zalpha11 ligand and the mutated hGH gene was obtained to be used for microinjection into fertilized murine oocytes.

Century construction for the expression of mouse zalpha11 ligand from the liver-specific promoter (MT-1

The same insert murine zalpha11 ligand from example 26A was subcloned into the vector pTG12-8, as described in example 22A. For this design, approximately 10 mg of makeprimary DNA FO51-zlh11-pigand were digested United Fsel and Asel, precipitated with ethanol and a fragment of a murine zalpha11 ligand was purified as described in example 22. Then this fragment is ligated into the vector pTG12-8, which was previously split Fsel and Asel, as described in example 22A. Electroporation, screening of clones and receiving makeprimary performed as described in example 22. Sall fragment containing the 5'- and 3'-flanking sequences, the MT promoter-1, insulin intron II of the rat cDNA of mouse zalpha11 ligand and a poly-a sequence hGH, received to be used for microinjection into fertilized murine oocytes.

Example 27

Polyclonal antibodies to murine zalpha11-ligand

Polyclonal antibodies were obtained by immunization of two female new Zealand white the rollers purified polypeptide muzalpha11/MBP-6H (example 32). Each rabbit received an initial intraperitoneal injection (ip) of 200 mg of purified protein in complete Freund's adjuvant followed booster ip injections of 100 mg of peptide in incomplete Freund's adjuvant every three weeks. After seven to ten days after the second booster injection (a total of 3 injections) produced krovoisliania animals and collect the serum. Then the rabbits continued to enter the booster injection and took blood every three weeks.

Muzalpha11L/MBP-6N-specific rabbit serum was previously adsorbing antibodies from anti-MBP using column CNBr-SEPHAROSE 4B protein a (Pharmacia LKB)that was prepared using 10 mg of purified recombinant malesurvivor protein (ICBMs) per gram CNBr-SEPHAROSE. Recombinant ICBM was obtained and was purified on amylose column in the authors laboratory using methods well known in the field, polyclonal muzalpha11-ligand-specific antibodies were affinity purified from rabbit serum using column CNBr-SEPHAROSE 4B protein a (Pharmacia LKB)that was prepared using 10 mg of purified recombinant protein specific antigen muzalpha11L/MBP-6H (example 32), followed 20X dialysis in TFR during the night. Muzalpha11-ligand-specific antibodies were characterized using ELISA using 1 µg/ml purified recombinant protein muzalpha11L-MBP-6H (example 32) or uzalpha11L-MBP-6H (example 32) as targets of antibodies. The lower threshold of detection (LLD) of the rabbit affinity purified antibody anti-muzalpha11L/MBP-6H is the breeding of 100 PG/ml on its specific purified recombinant antigen muzalpha11L/MBP-6H and 500 PG/ml on purified recombinant huzalpha11L-MBP-6H.

Example 28

Design xpressimage vector mammals and large-scale expression of zalpha11 ligand human cells Cho DG44

Expressing the vector mammals for zalpha11 ligand human (SEQ ID NO:1), is designed to add Sall site at the 5'-end and a Pmel site to the 3'-end cDNA was constructed by amplification using PCR from a plasmid containing zalpha11 ligand person (example 7) with oligonucleotide primers ZC22054 (SEQ ID NO:70) and ZC22055 (SEQ ID NO:71). Conditions for PCR reactions were as follows: 94°C for 4 minutes; 25 cycles of 94°C for 45 seconds, 50°C for 45 seconds and 72°C for 3 minutes; and 72°C for 10 minutes. The PCR product was isolated, as described here, and cut Sall and Pmel, then ligated into a plasmid pDC312, pre-cut to suitable restriction sites in polylinker, described using standard methods. Plasmid pDC312 described in Morris, A. et al., "Expression Augmenting Sequence Element (EASE) isolated from Chinese Hamster Ovary Cells", in Animal Cell Technology. Carrondo, MJT et al., (eds.) (1997) Kluwer Academic Publishers, The Netherlands, p.529-534.

Legirovannye vector was transfusional in adaptiva the data to suspension cells Cho DG44 (, in Novo Nordisk, Denmark) using lipofectin using reagent Lipofectamine™ (Gibco/BRL) according to the manufacturer's instructions. The transfectants were selected in medium PFCHO (JRH, Lenexa, Kansas), not containing thymidine and gipoksantina, with subsequent selection of 200 nm methotrexate (Sigma, St. Louis, MO). Resistant to methotrexate cell cloned breeding and testing for the production of zalpha11 ligand using activity analysis BaF3 (example 5B).

Productive clone were propagated and grown in 7-20-liter bioreactor (Applicon Bioreactors, Schiedam, The Netherlands) in the environment PFCHO with obtaining material for purification (example 29).

Example 29

Large-scale purification of unlabeled zalpha11 ligand of human and mouse expression of cell lines of mammalian KSS and SNO

A. Expressed SNO zalpha11 ligand person

Unless otherwise noted, all operations were carried out at 4°C. the Following procedure was used for the purification of zalpha11 ligand person of at least 30 liters SNO-conditioned medium (see example 28). Concentrated or non-concentrated conditioned medium (CC) sterile filtered through filters of 0.45 and 0.22 micron. Then a concentrated environment sauterelle 0.01 M MES (Fluka BioChemica, Switzerland)and the pH is brought to 6.0 and then applied to the column 50×100 mm (196 ml) Poros 50 HS (strong cation-exchanger from PerSeptive BioSystems, Framingham, MA) min is the night when 4-10 ml/min using a BioCAD SPRINT (Perceptive BioSystems). This column was washed with 10-20 column volumes (KO) 0.01 M MES/0,130 M NaCl (Mallinckrodt, Paris, KY) pH 6.0 Then associated proteins were suirable 10 TO gradient 0,130 M - 1 M NaCl in 0.01 M MES pH 6.0 at 30 ml/min; fractions of 25 ml were collected throughout chromatography and absorption at 280 and 215 nm were subjected to monitoring. Fraction peaks were analyzed using gel electrophoresis in SDS page-ordinator with silver staining (Geno Technology, St. Louis, MO), Kumasi (Sigma, St. Louis, MO) and Western Western blot turns using antibodies against zalpha11 ligand person (example 33 and example 34).

Fraction peaks were combined, then concentrated in the hub with a mixing cell on the membrane YM10 (Millipore/Amicon, Bedford, MA) to the regular level (1-10 ml). Then the sample was applied on a suitable gel-filtration column quick resolution (52-600 ml) Sephacryl S-200 (Pharmacia, Uppsala Sweden), equilibrated in SFR (Gibco BRL), at the speed of a current of 1-2 ml/min; fractions of 1-2 ml were collected throughout chromatography and absorption at 280 and 215 nm were subjected to monitoring. Fraction peaks were analyzed by gel-electrophoresis on SDS page-ordinator with silver staining (Geno Technology, St. Louis, MO) and Kumasi (Sigma, St. Louis, MO).

Interest fractions were combined and concentrated using centrifugal concentrators (Millipore 5 kD MWKO to the minimum volume. Then the final product was analyzed by gel electrophoresis on SDS page-the SN with staining of Kumasi (Sigma, St. Louis, MO), Western Western blot turns, N-terminal sequencing, amino acid analysis and BSA analysis (Pierce, Rockford, Illinois) to determine the purity and concentration of the protein.

Century Expressed KSS 570 murine zalpha11 ligand

Unless otherwise noted, all operations were carried out at 4°C. the Following procedure was used for the purification of zalpha11 ligand mouse from KSS-air-conditioned environment (example 18). Concentrated or non-concentrated conditioned medium (CC) sterile filtered through filters of 0.45 and 0.22 micron. Then Wednesday sauterelle 0.01 M MES (Fluka BioChemica, Switzerland)and the pH is brought to 6.0. This COP was analyzed, was applied on the column, and AS was suirable with a column as described in example 29A.

Interest fractions were combined, then concentrated in the hub with a mixing cell, as in example 29A, up to a volume of 20-30 ml the pH was brought to 7.0, then this sample was applied either on the column Poros AL 0.8 ml, which was about 3 mg zalpha11CFLAG-tagged soluble receptor (example 10B), or on the column with approximately 10 mg merged zalpha11-Fc4 receptor (example 10C)immobilized on the resin (see method below), at 1 ml/min on a BioCAD SPRINT. Then the column was washed with at least 20 TO 0.3 M NaCl/FR (Gibco BRL)/0.01 M MES at 10 ml/min and Then the column was quickly suirable injection 600 μl of 0.1 M glycine (Aminoacetic Add; Glycocol, Spectrum, Gardena, CA) pH of 2.5 at the speed of a current of 10 ml/m is n with PR on a BioCAD SPRINT. Fractions of 1 ml were collected for 6 seconds each, and immediately neutralized pH 55 μl of 2 M Tris pH 8.8 (Tris(hydroxymethyl)aminomethane, EM Science, Gibbstown, NJ). The absorption at 280 and 215 nm were monitored throughout the chromatography. Fraction peaks were analyzed using gel electrophoresis on SDS page-ordinator with silver staining (Geno Technology, St. Louis, MO), Kumasi (Sigma, St. Louis, MO) and Western Western blot turns, as described above.

Fraction peaks were combined, then concentrated in the hub with a mixing cell, as in example 29A to the minimum volume (1-10 ml). Then, the sample was applied, balanced and analyzed as in example 29A, on a suitable gel-filtration column quick resolution Sephacryl S-200 (Pharmacia). Fraction peaks were analyzed by gel-electrophoresis on SDS page-ordinator with silver staining (Geno Technology, St. Louis, MO) and Kumasi (Sigma, St. Louis, MO). Interest fractions were combined and concentrated and analyzed as in example 29A.

C. Immobilization of proteins on the media POROS AL

All surgeries were performed at room temperature on a BioCAD 700E. Column 4,5×50 mm flow was Packed by carrier POROS AL in 2 M NaCl in accordance with the descriptions of the manufacturer. Then the column was balanced in 1.1 M Na2SO4and 50 mm sodium phosphate pH of 7.2. The receptor was concentrated to 4 mg/ml using a centrifugal concentrator Mllipore 30 kD MWCO, then was diluted 1:1 in 1.1 M Na2SO4and 50 mm sodium phosphate pH of 7.2. Through the column was passed at 2 ml/min 1.1 M Na2SO4and 50 mm sodium phosphate pH 7.2 and was produced by injection of 100 μl of the diluted ligand every 9 TO, until reaching the stationary state of saturation or interruption through the column. Then passed 62 TO the gradient of 1.1 M Na2SO4and 50 mm Na-phosphate pH 7.2 to 550 mm Na2SO4and 50 mm Na-phosphate pH to 7.2 with 5 mg/ml cyanoborohydride sodium. The column was kept for 2 hours to complete immobilization. Then the column was balanced in 0.2 M TRIS pH to 7.2 with 5 mg/ml cyanoborohydride sodium and allowed to stand for about 1 hour. Finally the column was balanced in SFR from 0.02% sodium azide and stored at 4°s to use. Before using pre-column was suirable 0.1 M glycine for guarantees that were removed nonspecific proteins and that this column was not Vasilache immobilized receptor.

Example 30

Designing expressing vector, expression and purification of unlabeled zalpha11 ligand of human and mouse from baculovirus

A. Design for the expression of zalpha11 ligand and the man in the baculovirus

Expressing the vector, pzalpha11L, prepared for the expression of polypeptides zalpha11 ligand person in insect cells. Fragment 517 BP, containing the sequence zalpa11-ligand person and encoded restriction sites BamHI and Xhol on the 5'- and 3'-ends, respectively, generated PCR amplification from a plasmid containing cDNA zalpha11 ligand person (example 7) using primers ZC23444 (SEQ ID NO:74) and ZC23445 (SEQ ID NO:75). Conditions for PCR reactions were as follows: 1 cycle 94°C for 4 min; then 25 cycles at 94°C for 45 seconds, 50°C for 45 seconds and 72°C for 2 minutes; then 1 cycle at 72°C for 10 minutes; then soaking at 4°C. the Fragment was visualized using gel electrophoresis (1% SeaPlaque/1% NuSieve). The band was cut out, was diluted to 0.5% agarose 2 mm MgCl2, melted at 65°With, were digested BamHI and Xhol (Boehringer Mannheim) and ligated into the cleaved BamHI/Xhol expressing baculovirus vector, pZBV3L. Vector pZBV3L is a modification of the expressing vector pFastBac1™ (Life Technologies), where the promoter Poliakova was removed and replaced with late promoter activating basic protein. About 14 nanograms split restrictase inserts zalpha11 ligand and about 40 ng of the corresponding vector ligated overnight at 16°C.

Mixture for ligation was diluted 3 times in TE (10 mm Tris-HCl, pH 7.5 and 1 mm EDTA) and about 4 fmol of the diluted mixture for ligation was transformed into Library Efficiency competent cells DH5α (Life Technologies) according to the manufacturer's instructions by heat shock for 45 seconds in a water bath at 42°C. Transformieren the Yu DNA and cells were diluted in 450 μl of the environment SOC (2% Bacto™ Tripton, 0.5% Bacto-Yeast extract, 10 ml of 1 M NaCl, 1.5 mm KCl, 10 mm MgCl2, 10 mm MgSO4and 20 mm glucose) and were sown on LB-cups containing 100 μg/ml ampicillin. Clones were analyzed using the products of cleavage by restrictase and 1 µl of the positive clone was transformed into 20 μl D10 Max Efficiency competent cells (GIBCO-BRL, Gaithesburg, MD) according to the manufacturer's instruction, by heat shock as described above. Then the transformed cells were diluted in 980 μl of the environment SOC (2% Bacto™ Tipton, 0.5% Bacto-Yeast extract, 10 ml of 1 M NaCl, 1.5 mm KCl, 10 mm MgCl2, 10 mm MgSO4and 20 mm glucose), were grown in shake a thermostat at 37°C for 4 hours and sown on plates with Luria agar containing 50 μg/ml kanamycin, 7 ág/ml gentamicin (Life Technologies), 10 μg/ml tetracycline, IPTG (Pharmacia Biotech) and Bluo-Gal (Life Technologies). Seeded cells were incubated for 48 hours at 37°C. the Selection color used to identify cells with donor insert encoding zalpha11 ligand man, which was included in the plasmid (called "bacmids"). Colonies that had a white color, viscerale for analysis. DNA backside zalpha11 ligand person was isolated from positive colonies using the QiaVac Miniprep8 (Qiagen) according to the manufacturer's instructions. Clones were screened for correct ant the RT DNA amplification using primers to transposition element in backside by applying PCR primers ZC447 (SEQ ID NO:76) and ZC976 (SEQ ID NO:77). Conditions for PCR reactions were as follows: 35 cycles of 94°C for 45 seconds, 50°C for 45 seconds and 72°C for 5 minutes; 1 cycle at 72°C for 10 minutes; then soaking at 4°C. the PCR product was subjected to electrophoresis on 1% agarose to check the size of the inserts. Clones with the correct insert was used for transfection of cells Spodoptera frugiperda (Sf9).

C. Expression and to generate material for cleaning zalpha11 ligand person from baculovirus

The Sf9 cells were sown at 5×106cells per 35 mm Cup and allowed to attach for 1 hour at 27°C. Five microlitres backmenu DNA zalpha11 ligand person (described above) was diluted in 100 ál of medium Sf9-900 II SFM (Life Technologies). Six μl of CellFECTIN reagent (Life Technologies) was diluted with 100 μl Sf-900 II SFM. Backmenu DNA and solutions of lipids were carefully mixed and incubated for 30-45 minutes at room temperature. The environment of one Cup was aspirated, cells were washed 1×2 ml of fresh medium Sf-900 II SFM. 800 microliters Sf-900 II SFM was added to a mixture of lipid-DNA. The wash medium was removed by aspiration and the mixture of DNA-lipid was added to the cells. Cells were incubated at 27°C for 4-5 hours. The mixture of DNA-lipid was removed by aspiration, and 2 ml of medium Sf-900 II was added to each Cup. The cups were incubated at 27°C, 90% humidity for 96 hours after which the virus was collected.

For the primary amplification CL the TCI Sf9 were grown in 50 ml of medium Sf-900 II SFM in shake flask 125 ml to approximate the density of 0.41-0,52 × 105cells/ml the cells are Then infected with 150 ál of viral solution described above and incubated at 27°C for 3 days, after which the virus was collected in accordance with standard methods known in this field. A sample of 500 μl was used to determine activity in F3-test (example 5) to confirm that it was biologically active.

For secondary amplification of Sf9 cells were grown in 1 l medium Sf-900 II SFM in a shake flask at 2800 ml to approximate density of 0.5×105cells/ml was infected with 500 µl primary source of virus solution described above and incubated at 27°C for 4 days, after which the virus was collected in accordance with standard methods known in this field. The virus was titrated and grew to large scale purification produced by baculovirus zalpha11 ligand person (huzalpha11L-Bv), as described in example 30 and example 30D below.

C. large-Scale purification of expressed by baculovirus zalpha11 ligand human/mouse

Unless otherwise noted, all operations were carried out at 4°C. the Following procedure was used for the purification of zalpha11 ligand person (huzalpha11L-Bv) BV-air-conditioned environment (example 30B). Air-conditioned environment (CC) sterile filtered through filters of 0.45 and 0.22 micron, then sauterelle 0.01 M MES (Fluka Biohemica, Switzerland)and the pH is brought to 6.0. Then the COP was applied on the column POROS 50 HS and subjected to chromatography, fractions were collected, analyzed as described in example 29A.

The fractions obtained peaks were pooled, concentrated, subjected to chromatography on a gel filtration column high resolution and analyzed as described in example 29A.

Interest fraction from the gel filtration column were combined and concentrated centrifugal concentrators 5 KD MWCO Millipore to the minimum volume. Then the final product was analyzed by gel electrophoresis on SDS page-ordinator with staining of Kumasi (Sigma, St. Louis, MO), Western Western blot turns, N-terminal sequencing, amino acid analysis and BSA analysis (Pierce, Rockford, Illinois) to determine the purity and concentration of the protein, as described in example 29A. The whole protein was stored at -80°C.

R. Purification on a small scale (<2 mg) expressed by baculovirus zalpha11 ligand human/mouse

Unless otherwise noted, all operations were carried out at 4°C. the Following procedure was used for the purification of <2 mg zalpha11 ligand human or mouse from BV-air-conditioned environment. Air-conditioned environment (COP) was filtered, sauterelle and pH were made as in example 30. Then the COP was applied on the column, was suirable and chromatography POROS 50 HS were analyzed as described in example 30C.

Fractional, then was concentrated by diafiltration in the hub with a mixing cell on the membrane YM10 (10 KD MWCO (clipping mol mass) (Millipore/Amicon, Bedford, MA) until only a small amount (20-30 ml). the pH was brought to 7.0, and then the sample was applied either on the column, 0.8 ml Poros AL, which had about 3 mg zalpha11CFLAG-tagged soluble receptor (example 10B), or on the column with approximately 10 mg merged zalpha11-Fc4 soluble receptor (example 10C)immobilized on the resin (see method in example 29S), at 1 ml/min on a BioCAD SPRINT. Then the column was washed with at least 20 TO 0.3 M NaCl/FR (Gibco BRL)/0.01 M MES at 10 ml/min and Then the column was quickly suirable injection 600 μl of 0.1 M glycine (Aminoacetic Acid; Glycocol, Spectrum, Gardena, CA) pH of 2.5 at the speed of a current of 10 ml/min with FR on a BioCAD SPRINT. Fractions of 1 ml were collected for 6 seconds each, and immediately neutralized to pH 55 μl of 2 M Tris pH 8,8 (Tris(hydroxymethyl)aminomethane, EM Science, Gibbstown, NJ). The absorption at 280 and 215 nm were monitored throughout the chromatography. Fractions were analyzed as described above.

Fraction peaks were combined, then concentrated by diafiltration in the hub with a mixing cell on the membrane YM10 (10 KD MWCO (clipping mol mass) (Millipore/Amicon, Bedford, MA) to 1-2 ml Then, the sample was applied on a suitable gel-filtration column quick resolution Sephacryl S-200 (Pharmacia, Uppsala, Sweden), equilibrated in FR (Gibco BRL), at the optimum when Oreste current; fractions were collected throughout chromatography and absorption at 280 and 215 nm were subjected to monitoring. Fractions were analyzed as described above.

Interest fractions were combined, then concentrated centrifugal concentrators 5 KD MWCO Millipore to an insignificant amount. Then the final product was analyzed by gel electrophoresis on SDS page-ordinator with staining of Kumasi (Sigma, St. Louis, MO), Western Western blot turns, N-terminal sequencing, amino acid analysis and BSA analysis (Pierce, Rockford, Illinois) to determine the purity and concentration of the protein. The whole protein was stored as described above.

E. construction for the expression of mouse zalpha11 ligand in baculovirus: pzalpha11lig.M

Prepared expressing vector, pzalpha11LM, for the expression of polypeptides of the mouse zalpha11 ligand in insect cells. Fragment 413 P.K., containing the sequence for murine zalpha11 ligand and encoded restriction sites BspEl and Xbal at the 5'- and 3'-ends, respectively, generated PCR amplification from a plasmid containing cDNA of mouse zalpha11 ligand (example 16)using primers ZC25970 (SEQ ID NO:109) and ZC25969 (SEQ ID NO:110) using PCR-system high precision (Expand High Fidelity PCR System, Boehringer Mannheim) according to the manufacturer's instructions. The PCR conditions were as follows: 1 cycle 94°C for 2 minutes followed by 35 cycles of 94°C for 15 CE is und, 50°C for 30 seconds, and 72°C for 2 minutes; 1 cycle at 72°C for 10 minutes, followed by soaking at 4°C. a Small part of this PCR product was visualized by gel-electrophoresis (1% NuSieve agarose). The rest of this fragment was purified using the kit for purification of PCR product from Qiagen according to the manufacturer's instructions and was suirable in 30 μl of H2O. Then, this fragment was digested with restrictase Bspel and Xbal (Boehringer Mannheim) at 37°C for approximately 2 hours, and then subjected to electrophoresis on agarose as described above. The band was cut out, purified and suirable using the kit for the extraction of gels Qiagen according to the manufacturer's instructions. The purified fragment ligated in split Bspel/Xbal baculovirus expressing vector, pZBV37L. Vector pZBV37L is a modification of the expressing vector pFastBac1™ (Life Technologies), where the promoter Poliakova was removed and replaced with late promoter activating basic protein with subsequent secretory signal sequence from ecdysteroid-UDF-glucosyltransferase (EGT). About 5 µl split restrictase inserts murine zalpha11 ligand and about 100 ng appropriately cut vector ligated overnight at 16°With approximately 20 μl. Five µl of the mixture for ligation electropositive and 50 μl D12S-elecromagnetic bacterial cells, Life Technologies, using a 2 mm cuvette with the installation of 2 kV, 25 μf and 400 Ohms. Elektrooborudovanie the cells were placed in 1 ml of medium SOC (2% Bacto™ Tipton, 0.5% Bacto-Yeast extract, 10 ml of 1 M NaCl, 1.5 mm KCl, 10 mm MgCl2, 10 mm MgSO4and 20 mm glucose (Gibco BRL)), were grown at 37°C for approximately 1 hour and were sown on LB-cups containing 100 μg/ml ampicillin. DNA from these clones was isolated and analyzed using products by restriction cleavage to identify positive clones. Approximately 5 ng of DNA from a positive clone was transformed into 20 μl of DH10Bac Max Efficiency competent cells (GIBCO-BRL, Gaithesburg, MD) according to the manufacturer's instruction, by heat shock for 45 seconds in a water bath at 42°C. Then, the transformed cells were diluted and grown as described in example EVILS. Bacmid containing insert murine zalpha11 ligand identified and allocated as described in example 30A. Clones were screened for the correct insert by DNA amplification using primers to transposition element in this backside by PCR using primers ZC447 (SEQ ID NO:76) and ZC976 (SEQ ID NO:77). Conditions for PCR reactions were as follows: 35 cycles of 94°C for 45 seconds, 50°C for 45 seconds and 72°C for 5 minutes; 1 cycle at 72°C for 10 minutes; then soaking at 4° C. the PCR product was subjected to electrophoresis on 1% agarose to check the size of the inserts. Clones with the correct insert was used for transfection of cells Spodoptera frugiperda (Sf9).

F. Expression and generation of material for purification of mouse zalpha11 from baculovirus

The Sf9 cells were sown at 1 million cells per 35 mm Cup and allowed to attach for 1 hour at 27°C. Backmenu DNA zalpha11 ligand mouse was transfusional as described in example 30B, and the virus was collected.

For the primary amplification Sf9 cells were sown as described above, and 500 μl of the supernatant after 72 hours after transfection) was added and cultures were allowed to continue for 96 hours, after which the virus was collected in accordance with standard methods.

For secondary amplification of Sf9 cells were sown as described above, was added 200 μl of the primary source of virus solution. Cultures were incubated at 27°C for 72 hours, after which the virus was collected in accordance with standard methods.

For tertiary amplification, 10 µl of the amplified secondary source of virus solution was placed on Sf9 cells at 500,000 cells per well in 50 ml of SF900II medium in shake flask 250 ml for 6 days and the virus was harvested as described above. The virus was titrated and grew to large scale purification produced by baculovirus murine zalpha11-is Uganda (muzalpha11L-Bv), as described in example 30 and example 30D.

The presence of a protein with the predicted molecular weight in the supernatant was determined by Western analysis using polyclonal antibodies against muzalpha11L/MBP-6H (example 27). Analysis of proliferation test based VARZ (example 5) also showed that the secretory of liked was active.

Example 31

Expression of zalpha11 ligand of human and mouse in E. coli

A. Construction of vector star/Harpa-ligand expressing the fusion zalpha11 ligand man-MBP

The expression plasmid containing polynucleotide encoding zalpha11 ligand person, fused to the N-end with malesurvivor protein (MBP), designed by homologous recombination. The cDNA fragment zalpha11 ligand human (SEQ ID NO:1) was isolated using PCR. Two primers were used in obtaining fragment zalpha11 ligand person in a PCR reaction: (1) Primer ZC22128 (SEQ ID NO:78), containing 40 BP of the vector flanking sequence and 26 BP corresponding to the amino-end of the zalpha11 ligand person, and (2) primer ZC22127 (SEQ ID NO:79), containing 40 BP of the 3'-end corresponding to the flanking vector sequence, and 28 BP, corresponding carboxyl-end of the zalpha11 ligand person. Conditions for PCR reactions were as follows: 25 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 1 minute, followed by soaking PR is 4° C, the reaction was carried out in two replications. Two μl of the 100 μl PCR reaction was subjected to electrophoresis on a 1.0% agarose gel with 1 x TBE-buffer for analysis and observed the expected band of approximately 472 P.N. Remaining 90 μl of PCR reaction was combined with the second PCR tube, besieged 400 μl of absolute ethanol to be used for recombination into the Smal cut recipient vector rtar to obtain designs that encodes a fused polypeptide MBP-zalpha11 ligand, as described below.

Plasmid rtar was obtained from the plasmid pRS316 and pMAL-c2. Plasmid pRS316 is a Shuttle vector for Saccharomyces cerevisiae (Hieter P. and Sikorski, R., Genetics 122:19-27, 1989). pMAL-C2 (NEB) is the expression plasmid of E. coli. She is the tac promoter, triggering MalE (the gene encoding MBP)followed by a His-tag, thrombin cleavage, the cloning site and the rrnB terminator. Vector rtar designed using homologous recombination in yeast. 100 ng of EcoRI cut pMAL-c2 recombinable with 1 µg Pvul cut pRS316, 1 μg linker, and 1 μg cut Scat/EcoRI pRS316. The linker consisted of oligonucleotides ZC19372 (SEQ ID NO:80) (100 pmol): ZC19351 (SEQ ID NO:81) (1 pmol): ZC19352 (SEQ ID NO:82) (1 pmol) and ZC19371 (SEQ ID NO:83) (100 pmol), combined in a PCR reaction. Conditions for PCR reactions were as follows: 10 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 30 seconds; then soaking at 4°C. PCR-p the FL was concentrated by precipitation with 100% ethanol.

100 μl of competent yeast cells (S. cerevisiae) were combined with 10 µl containing approximately 1 μg of PCR product zalpha11 ligand person and 100 ng of Smal split vector star, and transferred into a cuvette for electroporation see 0,2 Mix the yeast/DNA was subjected to electric shocks at 0.75 kV (5 kV/cm), "undefined" number of Ohms, 25 μf. To each cuvette was added 600 μl of 1.2 M sorbitol and the yeast sown in two aliquot of 300 μl on two cups of URA-D and incubated at 30°C.

After approximately 48 hours Ura+ yeast transformants from a single Cup resuspendable in 1 ml of H2O and Unscrew the fast sedimentation of yeast cells. Cellular precipitate resuspendable in 1 ml lisanova buffer (2% Triton X-100, 1% LTOs, 100 mm NaCl, 10 mm Tris, pH 8.0, 1 mm EDTA). 500 ál lizinoj mixture was added to the Eppendorf tube containing 300 μl acid washed glass granules and 200 ál of a mixture of phenol/chloroform was shaken on a vortex two or three times at intervals of 1 min, with subsequent 5-minute loosening in the Eppendorf centrifuge at maximum speed. Three hundred microlitres the aqueous phase was transferred into a fresh tube and DNA was besieged by 600 ál of ethanol (EtOH), followed by centrifugation for 10 minutes at 4°C. the precipitated DNA resuspendable in 100 μl of N2O.

Transformation electrocompetent the cells of E. coli (MC1061, Casadaban et al., J. Mol. Biol. 138, 179-207) conducted the with 1 μl of a preparation of yeast DNA and 40 μl of MC1061 cells. Cells were subjected to electrical pulses at 2.0 kV, 25 MF and 400 Ohms. After electroporation, 0.6 ml SOC (2% Bacto™ Tripton (Difco, Detroit, Ml), 0.5% of yeast extract (Difco), 10 mm NaCl, 2.5 mm KCl, 10 mm MgCl2, 10 mm MgSO4, 20 mm glucose) were sown in a single aliquot of the Cup with LB AMR (LB-broth (Lennox), 1.8% Bacto™ agar (Difco), 100 mg/l ampicillin).

Individual clones carrying the correct expression construct for zalpha11 ligand man, identified by expression. Cells were grown in Superbroth II (Becton Dickinson) with 100 μg/ml ampicillin overnight. 50 µl of the overnight culture was used for inoculation of 2 ml of fresh Superbroth II + 100 μg/ml ampicillin. The cultures were grown at 37°With shaking for 2 hours. 1 ml of this culture was induced 1 mm IPTG. After 2-4 hours 250 μl of each culture was mixed with 250 μl acid washed glass pellets and 250 ál of buffer Turner with 5% β-me and dye (8 M urea, 100 mm Tris pH 7.0, 10% glycerol, 2 mm EDTA, 5% LTOs). The samples were shaken on a vortex for one minute and was heated to 65°C for 5-10 minutes. 20 μl was applied to a track on a 4-12% SDS page electrophoresis (NOVEX). Separation on gels occurred in the buffer 1xMES. Positive clones were named star and they were subjected to sequencing analysis. Polynucleotide sequence of the fused protein MBP-zalpha11 ligand person shown in SEQ ID NO:84, and sootvetstvuyushiye in SEQ ID NO:85.

C. Bacterial expression of zalpha11 ligand person

One µl of DNA sequencing was used for transformation of strain W3110 (ATSS). Cells were subjected to electrical pulses at 2.0 kV, 25 μf and 400 Ohms. After electroporation was added 0.6 ml SOC (2% Bacto™ Tripton (Difco, Detroit, Ml), 0.5% Of Yeast extract (Difco), 10 ml NaCl, 2.5 mm KCl, 10 mm MgCl2, 10 mm MgSO4and 20 mm glucose) were sown in a single aliquot of the Cup LB AMR (LB-broth (Lennox), 1.8% Bacto™ agar (Difco), 100 mg/l ampicillin).

Individual clones expressed. Cells were grown in Superbroth II (Becton Dickinson) with 100 μg/ml ampicillin overnight. 50 µl of the overnight culture was used for inoculation of 2 ml of fresh Superbroth II + 100 μg/ml ampicillin. The cultures were grown at 37°With shaking for 2 hours. 1 ml of this culture was induced 1 mm IPTG. After 2-4 hours 250 μl of each culture was mixed with 250 μl acid washed glass pellets and 250 ál of buffer Turner with 5% β-me and dye (8 M urea, 100 mm Tris pH 7.0, 10% glycerol, 2 mm EDTA, 5% LTOs). The samples were shaken on a vortex for one minute and was heated to 65°C for 10 minutes. 20 μl was applied to a track on a 4-12% SDS page gel electrophoresis (NOVEX). Separation on gels occurred in the buffer 1xMES. Positive clones were used for cultivation for cleaning the slit protein huzalpha11L/MBP-6H (example 32 below).

C. Design vector is TAR/zlh11-pigand mouse expressing the fusion zalpha11 ligand mouse/MBR

The expression plasmid containing polynucleotide encoding part of the zalpha11 ligand mouse, merged to M-end with malesurvivor protein (MBP), designed by homologous recombination as described in example 31A. The cDNA fragment zalpha11 ligand mouse (SEQ ID NO:55) was isolated using PCR. Two primers were used in obtaining fragment zalpha11 ligand mouse in a PCR reaction: (1) Primer ZC22849 (SEQ ID NO:86), containing 40 BP of the vector flanking sequence and 24 BP corresponding to the amino-end of the zalpha11 ligand mouse, and (2) primer ZC22850 (SEQ ID NO:87), containing 40 BP of the 3'-end corresponding to the flanking vector sequence, and 21 BP corresponding to carboxyl-end of the zalpha11 ligand mouse. The conditions of PCR reaction were the same as described above. A fragment of approximately 450 BP was cloned in RTR, as described above. Clones transformed, identified and cultivated as described above. Positive clones were named star and they were subjected to sequencing analysis. Polynucleotide sequence of the fused protein ICBM-mouse zalpha11 ligand in RTR shown in SEQ ID NO:88, and the corresponding polypeptide sequence is shown in SEQ ID NO:89. Positive clones were used for cultivation in E. coli as described above for purification of fused protein muzalpha11L/MBP-6H (example 32).

Por the measures 32

Cleaning zalpha11-MBP-ligand or zalphall-MBP-receptor

Unless otherwise noted, all operations were carried out at 4°C. the Following procedure was used for purification of mergers zalpha11-MBP-ligand for human zalpha11-MBP-ligand (huzalpha11L/MBP-6H) or murine zalpha11-MBP-ligand (muzalpha11L/MBP-6H) of E. coli. Fused proteins of human or murine zalpha11-MBP-receptor was obtained using the same method. Previously loose frozen E. coli paste was thawed and diluted in 2 liters of buffer (0.02 M TRIS (EM Science); 0.2 M NaCl (Mallincrodt); 0.01 M 2-mercaptoethanol (EM Science); pH 8.0; 5 mg/l pepstatin A (Boehringer Mannheim); 5 mg/l Aprotinin (Boehringer Mannheim) and 1 mg/l PMSF (Fluka)) plus 1-2 ml protivovspenivayushchie reagent AF289 (Sigma). The mixture was processed in a pre-cooled destroyer cells French Press (Constant Systems LTD) with 20-30 KF/square inch.

Then the lysate was centrifuged at 18000 × g for 45 minutes at 4°C; the supernatant was retained. 200 ml of a suspension of Amylose resin (New England BioLabs), pre-equilibrated in buffer A (0.02 M TRIS (EM Science); 0.2 M NaCl (Mallincrodt); 0.01 M 2-mercaptoethanol (EM Science); pH 8.0)was added to the supernatant of the lysate and incubated overnight in roller bottles 2 l for maximum absorption fused MBP-protein in the boot of the mixture. The resin was washed in the format of the boot column 5 column volumes of buffer A, then the download was suirable buffer C (buffer a with 0,0-maltose (Sigma)). The crude fractions were collected and subjected to monitoring by absorption at 280 nm.

Suirvey protein was analyzed by electrophoresis SDS NuPAGE (NOVEX) with staining of Kumasi (Sigma). The sample and the entire volume of the protein was stored at -80°C.

Example 33

Polyclonal antibodies to zalpha11-ligand person

Polyclonal antibodies to zalpha11-ligand person was obtained by immunization of two female new Zealand white rabbits with purified polypeptide huzalpha11L/MBP-6H (example 32) or purified Cho recombinant protein huzalpha11L-CHO (example 29). Each rabbit received an initial intraperitoneal injection (ip) of 200 mg of purified protein in complete Freund's adjuvant followed booster ip injections of 100 mg of purified protein in incomplete Freund's adjuvant every three weeks. After seven to ten days after the second booster injection (a total of three injections) produced krovoisliania animals and collect the serum. Then the rabbits continued to enter the booster injection and took blood every three weeks.

Rabbit serum induced to huzalpha11L/MBP-6H, previously had adsorbing antibodies against ICBMs using column CNBr-SEPHAROSE 4B protein a (Pharmacia LKB)that was prepared using 10 mg of purified recombinant MBP per gram CNBr-SEPHAROSE (Pharmacia). Recombinant MBP was prepared and purified on amylose column in the laboratory using the m ways well known in this field. Huzalpha11-ligand-specific polyclonal antibodies were affinity purified from the serum of rabbits using column CNBr-SEPHAROSE 4B protein a (Pharmacia LKB)that was prepared using 10 mg of the specific antigen purified recombinant protein huzalpha11L/MBP-6H or 10 mg of purified Cho recombinant protein huzalpha11L-CHO per gram CNBr-SEPHAROSE, followed 20X dialysis in FR during the night. Huzalpha11-ligand-specific antibodies were characterized using ELISA using 1 µg/ml purified recombinant proteins huzalpha11L/MBP-6H (example 32), zalpha11 ligand person (huzalpha11L-CHO) (example 29) or muzalpha11L-MBP/6H (example 32) as targets of antibodies.

The lower threshold of detection (LLD) of the rabbit affinity purified antibody anti-huzlh11/MBP-6N was breeding 10 PG/ml on its specific purified recombinant antigen huzalpha11L/MBP-6H, 500 PG/ml on purified recombinant huzalpha11L-CHO and 100 PG/ml on purified rekombinantnog muzalpha11L-MBP/6H (example 32). LLD rabbit affinity purified antibody anti-huzalpha11L-CHO was the breeding of 20 PG/ml on its specific purified recombinant antigen huzalpha11L-CHO, 500 PG/ml on purified recombinant huzalpha11L/MBP-6H and 50 ng/ml on purified recombinant muzalphal 1L/MBP-6H.

Example 34

Polyclonal antibodies to peptide zalpha11 ligand person

Polyclonal antibodies to peptide zalpha11 ligand human is and was obtained by immunization of two female new Zealand white rabbits with the peptide zalpha11 ligand person, huzalpha11L-1 (SEQ ID NO:72) or huzalpha11L-3 (SEQ ID NO:73). These peptides were synthesized using a peptide synthesizer Applied Biosystems Model 431A (Applied Biosystems, Inc., Foster City, CA) according to the manufacturer's instructions. Then the peptides conjugatively with protein carrier by hemocyanine fissurella (KLH) - activated maleimido. Each rabbit received an initial intraperitoneal injection (ip) of 200 mg of purified protein in complete Freund's adjuvant followed booster ip injections of 100 mg of purified protein in incomplete Freund's adjuvant every three weeks. After seven to ten days after the second booster injection (a total of three injections) produced krovoisliania animals and collect the serum. Then the rabbits continued to enter the booster injection and took blood every three weeks.

Serum of rabbits induced by the peptides zalpha11 ligand person, characterized by checking the titer ELISA using 1 μg/ml of the appropriate peptide used to produce the antibody (SEQ ID NO:72 or SEQ ID NO:73) as a target antibody. Two rabbit serum to the peptide huzalpha11L-1 had a titer in relation to their specific peptide at a dilution of 1:5000000 (1:5×106). Two rabbit serum to the peptide huzalpha11L-3 had the title in relation to their specific peptide at a dilution of 1:5×106.

Polyclonal antibodies specific for the peptide zalpha11-Li gang is a man, was affinity purified from rabbit serum using column CNBr-SEPHAROSE 4B protein a (Pharmacia LKB)that was prepared using 10 mg of the corresponding specific peptide (SEQ ID NO:72 or SEQ ID NO:73) per gram CNBr-SEPHAROSE, followed 20X dialysis in FR during the night. Huzalpha11-ligand-specific antibodies were characterized by scanning titer using ELISA using 1 μg/ml of the appropriate purified peptide antigen or purified recombinant full-sized proteins as targets of antibodies.

The lower threshold of detection (LLD) of the rabbit affinity purified antibody anti-huzalpha11L-l is the breeding of 500 PG/ml on its specific peptide antigen (huzalpha11L-1; SEQ ID NO:72), 500 PG/ml on purified recombinant huzalpha11L/MBP-6H (example 32) and 500 PG/ml on purified SNO-recombinant huzalpha11L-CHO (example 29). Not observed cross-reactivity relative to purified recombinant muzalpha11L/MBP-6H (example 32). LLD rabbit affinity purified antibody anti-Huzlh11-3 equal to 50 PG/ml on its specific peptide antigen (huzalpha11L-3; SEQ ID NO:73), 50 PG/ml on purified SNO-recombinant huzalpha11L-CHO (example 29) and 100 PG/ml on purified recombinant baculovirus huzalpha11L-Bv (example 30). Observed cross-reactivity against purified muzalpha11L/MBP-6H (example 32) with LLD 5 ng/ml.

Example 35

Monoclonal antibodies to zalpha11-receptor human

Monoclonal antibodies to zalpha11-receptor was obtained by immunization of 5 BALB/c mice (Harlan Sprague-Dawley, Indianapolis, IN) recombinant purified protein soluble receptor, zalpha11CEE (huzalpha11-CEE-BHK) (example 10A). Each mouse received an initial intraperitoneal (ip) injection of 20 mg of purified protein in complete Freund's adjuvant (Pierce, Rockford, IL) with subsequent booster ip injections of 10 mg of purified protein in incomplete Freund's adjuvant every two weeks. After seven to ten days after the introduction of the third booster injection was made krovoisliania animals and collect the serum.

Samples of sera of mice induced to huzalpha11-CEE-BHK, characterized by checking the titer using ELISA using purified recombinant Cho huzalpha11-Fc protein (example 10C) as a target antibody. Sample one mouse serum had a titer against specific target antibody at a dilution of 1:1,000,000 (1:1×106). Sample four mouse sera had titers against specific target antibody at a dilution of 1:100000 (1:1×106).

Splenocytes were collected from 4 mice with high titers and was merged with cells of the mouse myeloma SP2/0 using PEG 1500 (Boehringer Mannheim, UK) in two separate procedures merge with the application of merge 4:1 splenocytes to myeloma cells (Antibodies: A Laboratory Manual. E. Harlow and D. Lane, Cold Spring Harbor Press). After 10 days of growth after the merger clause is obserwuj specific antibodies of hybridoma identified using ELISA using purified recombinant KSS protein human zalpha11-RS (example 10C) as a target antibody and using FACS using cells BaFS expressing the sequence huzalpha11 (example 4 and example 2), as the target antibodies. Received 4 hybridoma, positive according to both methods, cloned three times by limiting dilution. Antibodies were identified: 249.28.2.1.2.2; 247.10.2.15.4.6; 249.19.2.2.3.5 and 249.15.2.4.2.7.

Example 36

Zalpha11 ligand-transgenic mouse

A. Obtaining transgenic mice expressing zalpha11 ligand of human and mouse

DNA fragments from transgenic vectors (example 22 and example 26), containing the 5' and 3' flanking sequences of the corresponding promoter (liver-specific promoter (MT-1 (zalpha11 ligand mouse (example 26C) or lymphoid-specific LCK promoter (zalpha11 ligand mouse and human (examples 26A and 22B), insulin intron II of the rat cDNA zalpha11 ligand and poly A-sequence of human growth hormone, received and used for microinjection into fertilized murine oocytes B6C3f1 (Taconic, Germantown, NY) using a standard Protocol microinjection. Cm. Nedap, C. et al., Manipulating the Mouse Embyio. A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1994.

Eight transgenic mice expressing zalpha11 ligand man from lymphoid-specific promoter EμLCK identified among the 44 calves. Four of these calves died, and 4 grew to adulthood. The expression levels were clearly lower in these animals. Twenty transgenic mice, xpressway murine zalpha11 ligand from lymphoid-specific promoter Eμ LCK identified among 77 calves. All 20 grew to adulthood. The expression levels were clearly lower in these animals. Three transgenic mice expressing mouse zalpha11 ligand from the liver-specific promoter (MT-1, was identified among the 60 calves. Two of these calves died, and 1 grew to adulthood. The expression levels were clearly lower in these animals. Tissues were histologically prepared and examined as described below.

C. Microscopic evaluation of tissues of transgenic mice

Spleen, thymus and mesenteric lymph nodes were collected and analyzed for histological studies of transgenic animals expressing zalpha11 ligand mouse and human (example 36A). Other tissues that were collected routine, include the following tissues: liver, heart, lung, kidney, skin, mammary gland, pancreas, stomach, small intestine, large intestine, brain, salivary gland, trachea, esophagus, adrenal gland, pituitary gland, sexual way, an extension of the male sex glands, skeletal muscle, including peripheral nerve, femur with bone marrow. These tissues were collected from neonatal calves that died unexpectedly, and several adult transgenic mice, as described below. Samples were fixed in 10% buffered formalin, processed routinely, alibali in paraffin, did slices at 5 microns and stained them with hematoxylin and eosin. Slides were examined and evaluated in points in relation to the severity of tissue changes (0=no change, 1=slight change, 2=moderate, 3=severe) veterinary pathologist with a certificate of the Ministry, not knowledgeable about this or that treatment.

Calf and 2 females adult mice expressing zalpha11 ligand person, and 3 of 6 male adult mice expressing zalpha11 ligand mice showed inflammatory infiltrates in many of the tested tissues. Affected organs varied to some extent from mouse to mouse. The inflammatory infiltrate consisted mainly of neutrophils and macrophages in varying quantities and proportions and had a weak - moderate in severity. In addition, these animals have found changes in lymphoid organs, including moderate to severe lymphopenia in the spleen and thymus (transgenes zalpha11 ligand of human and mouse); and heavy lymphopenia (transgenes zalpha11 ligand person) or mild to severe abdominal pyogranulomatous lymphadenitis (transgene zalpha11 ligand mouse) in the lymph nodes. In addition, the increased famedollars haematopoiesis was evident in the spleens. These changes were not observed in the control mice of the same age.

C. Flowing cytometrics analysis of TKA is it from transgenic mice sverkhekspressiya zalpha11 ligand

Transgenic animals, sverkhekspressiya zalpha11 ligand man or mouse (example 36A), was slaughtered for flow cytometrical analysis of peripheral blood, thymus (thymus), lymph node, bone marrow and spleen.

Cell suspensions were prepared from spleen, thymus and lymph nodes by the tearing of the body of the tweezers into a chilled on ice culture medium (500 ml medium RPMI 1640 (JRH Biosciences, Lenexa, KS), 5 ml 100x L-glutamine (Gibco BRL, Grand Island, NY); 5 ml 100x Na-pyruvate (Gibco BRL), 5 ml 100x penicillin, streptomycin, neomycin (PSN) (Gibco BRL) and then gently crushed these cells through the cell deformer (Falcon, VWR Seattle, WA). Peripheral blood (200 ml) was collected in heparinized tubes and diluted to 10 ml HBSS containing 10 E of heparin/ml of Erythrocytes were removed from the preparations of spleen and peripheral blood hypotonic lysis. Suspensions of bone marrow were prepared by leaching of bone marrow from the femurs chilled on ice cultural environment. The cells were counted and tested for viability using Trypanosoma blue (GIBCO BRL, Gaithesburg, MD). Cells resuspendable in chilled on ice coloring medium (HBSS, 1% fetal calf serum, 0.1% of sodium azide) at a concentration of ten million per milliliter. Blocking of Fc-receptor and non-specific is wyzwania antibodies to the cells was achieved by adding 10% normal goat serum and Fc block (Pharmingen, La Jolla, CA) to cell suspension.

The cell suspension was mixed with equal volumes of labeled fluorochroman monoclonal antibodies (PharMingen), incubated on ice for 60 minutes and then washed twice in chilled on ice proryvnym buffer (STR, 1% fetal calf serum, 0.1% of sodium azide) before resuspending 400 ml wash buffer, containing 1 mg/ml 7-AAD (Molecular Probes, Eugene, OR) as a marker of viability in some samples. Flow data were obtained on a flow cytometer FACSCaIibur (BD Immunocytometry Systems, San Jose, CA). Both receiving and analysis were performed using CellQuest software (BD Immunocytometry Systems).

Transgenic animals that expressed zalpha11 ligand human or mouse with high levels had dramatically altered the population of cells in all lymphoid organs analyzed. The observed changes included a complete loss of saturation of the cells of the thymus, the complete absence of D45R-positive b-cells and increased the size and density of cells in the spleens. As the spleen and bone marrow were increased numbers of myeloid cells in size, which is caused by increases in the number of monocytes and neutrophils. Marker pan NK cells (DX5) has been increasing in many populations. Moderately xpressimage founders had less dramatic, but still significant changes, with glasses with phenotype observed in cells with high expression. Mice with the lowest expression level had no significant increase in myeloid cells, no decrease of the numbers In the cells. They indeed found significant changes in populations of thymocytes with decreases in CD4+CD8+ double-positive cells and increases in CD4+and CD8+ separately positive cells.

Example 37

The study of the dependence of the response, the dose-response protein a purified recombinant human zalpha11 ligand person in normal mice

A. the Essence of research

The normal six-week female mice C57B1/6 (Harlan Sprague-Dawley, Indianapolis, IN) were treated by intraperitoneal injection once a day for four or eight days in one of four dose levels of purified recombinant human zalpha11 ligand person (example 24) with of 0.1, 0.5, 5 or 50 μg/mouse/day or carrier as a control. Body weight and body temperature were monitored once a day. At day 4 or day 9, four of the eight mice from each group processing protein and five of the ten mice in the control group with the carrier squashed. Blood, bone marrow and tissues were collected and analyzed. Investigated potential violations in lymphoid tissues, and normal physiological and Toxicological parameters.

No evidence of toxicity of the protein zalpha11 ligand person when Liu the Oh of the tested doses. Body weight and body temperature were unchanged. No visible changes in clinical chemistry parameters. However, there were persistent facts related to an increased percentage of cells of the myeloid lineage differentiation in the bone marrow, spleen and peripheral blood in mice treated with the highest dose of zalpha11 ligand, in comparison with the control carrier. Observed statisticheski significant increase of cells size of cells of the myeloid lineage differentiation, identified flow-cytometrical analysis of spleen homogenate in the group with high dose. Spleen two groups with the highest dose were statistically significantly larger than the spleen of other groups. However, histopathological study, only a negligible increase famedollars haematopoiesis was observed in the group with the highest dose. A statistically significant increase was in respect of myeloid cells to the erythroid bone marrow in the group with the highest dose compared with other groups. Finally, the observed increase in the peripheral blood as numbers of total leukocytes and the percentage of monocytes in the same group.

C. Preparation of solutions for injection doses

Purified recombinant human zalpha11 ligand person (example 24) was diluted in sterile phosphate buffered saline (Gibco BRL, Grand Island, NY) at concentrations to deliver 50, 5, 0.5 or 0.1 micrograms of protein in 0.1 ml of carrier-STR. Dose for the first four days were prepared on day 0 and froze in the cold freezer at -20°s to use. Doses for days 5-8 were prepared on day 5 and frozen as described above. Aliquots of the same SFR frozen processed by the media control group. On the day of introduction of appropriate aliquots were thawed and 0.1 ml were injected with intraperitoneally into each mouse for either four or eight days.

C. Plan of study

Mice were six weeks at the beginning of this study. Each processing group consisted of eight mice with the exception of the control group with the medium, which consisted of ten mice. Half of the mice of each group were killed after four days of treatment, and the other half after eight days.

Before processing each day, each mouse was weighed and the temperature of her body were recorded using a portable system programmable laptop (BMDS Inc., Maywood, NJ) scanning of the mouse on the identification number and the body temperature of the implanted subcutaneously respondents (IPTT-100, BMDS, Maywood, NJ).

When the death of the tissue were collected to assess populations of leukocytes using flow cytometrical analysis, including bone marrow, thymus and spleen. FACS-analysis of lymphoid organs and bone marrow is ispolnali using FACSCalibur (Becton Dickinson, Mansfield, MA). Tissues collected for histological examination for signs of toxicity of the protein consisted of: spleen, thymus, liver, kidney, adrenal, heart, and lungs. All tissue fixed for histology, kept at 4°over night in 10% normal buffered saline solution (NBF) (Surgipath, Richmond, IL). The next day NBF was replaced by 70% ethanol and the tissue returned to conditions 4°before processing for histology.

Tissues were processed and stained with hematoxylin and eosin in the laboratory and then sent contracted to the pathologist for histological analysis. Blood was collected for clinical blood count (CBC) and indicators of the chemical profile of serum, SVS were analyzed in the laboratory using the analyzer Cell Dyn 3500 Hematology Analyzer (Abbott Diagnostics Division, Abbott Park, IL) and the manual leukocyte counts were analyzed in the laboratory Phoenix Central Laboratory (Everett, WA). Serum was kept frozen at -20°to provide in Phoenix Central Laboratory for complete panels chemical parameters serum. To assess the relationship myeloid : erythroid, bone marrow from one femur was done on CytoSpin slides (CYTOSPIN 3 CYTOCENTRIFUGE and CYTO SLIDES, Shandon, Pittsburgh, PA) and was sent to Phoenix Central Laboratory for analysis.

D. the results of the study

There was no apparent clinical guidance on the physical and the ideological effects or toxicity zalpha11 ligand person at doses of 50 mg/day or lower doses. Body weight and temperature remained normal during these treatments. Chemical indicators sera were within normal limits. The number of erythrocytes and platelets were apparently normal. In mice receiving 50 mg/day for 8 days, manual received leukocyte formula showed that the percentage of monocytes was increased in peripheral blood and there was a visible increase in the total number of leukocytes. In bone marrow, washed from the femur, myeloid-erythroid increased in the group with a dose of 50 mg and to a lesser extent in the group with a dose of 5 μg of 8-day-delivery doses. In nonparametric comparison of multiple columns using InStat (InStat MAC; GraphPad Software, Inc., San Diego, CA) this difference was statistically significant (p=0,0049). The difference between the group with the highest dose and a group of media was also significant (p=0,0286). Thus, the increased number of leukocytes in peripheral blood and a significant increase in myeloid precursors can be linked to each other.

Histological evaluation of tissues found no visible evidence of cytological or structural changes, mitotic events or necrosis: thymus, liver, kidney, adrenal gland, duodenum, pancreas, jejunum, cecum, colon, mesenteric lymphatic the ski nodes of the uterus, ovary, salivary glands, heart, trachea, lung and brain. There was no apparent difference between treatment groups in weight of the thymus, kidney, liver or brain. From all tissues tested had a significant effect only on the weight of the spleen.

The weight of the spleen of each mouse were normalized relative to the weight of its brain. In the processing group 50 µg/day in comparison with media groups processing of 0.1 μg and 0.5 μg, the average weight of the spleen were almost 50% more after four days of treatment and almost 100% more after eight days than the average weight of the spleen of the other three groups. The 4-day experiment group 5 µg/day also had an increase in spleens compared with the control group and the groups with low doses. The difference in the relationship of the weights of the spleen/brain with data from a 4-day and 8-day productions, combined with the processing group was statistically significant (p=0,0072) according separatrices ANOVA Kruskall-Wallace, the test comparing multiple columns using the program InStat (GraphPad Software).

A slight increase famedollars of hematopoiesis, in particular, in the red pulp, was evident in the spleens of mice in the group with the highest dose, even in mice treated for four days. Running cytometrics analysis of the spleens showed a significant increase in the proportion of myeloid cells size in the group with Eivissa dose (p=0.01, t-student test), representing the increase in both monocytes and neutrophils. This effect may be associated with an increased percentage of mononuclear cells in peripheral blood, but also a visible increase in myeloid precursors in the bone marrow, as described above. In addition, transgenic mice obtained by insertional gene zalpha11 man, had increased famedollars haematopoiesis in their spleens compared to necroshine odnopolnymi animals.

Several changes were observed in the group with a dose of 50 µg/day in comparison with the control group, which require the participation of zalpha11 ligand in the formation and development of cells of the myeloid lineage differentiation. Taken together, these observed changes suggest that zalpha11 can be used as a therapeutic protein in such medical applications as cancer and immune disorders described herein.

Example 38

A preliminary study of elimination and tissue distribution of the purified recombinant protein zalpha11 ligand person

A. the Essence of research

To clarify the nature of the tissue distribution and elimination cleaned rhzalpha11-ligand was undertaken preliminary pharmacokinetic study. Devyatisilnyi male mice C57B1/6 received purified recombinant protein zalpha11 ligand brow of the ESA, labeled111indium (111ln) (NEN, Boston, MA) in one of three ways. Each mouse received a single bolus intravenous injection (iv), intraperitoneal (ip) or subcutaneous route (sc). Mice injected subcutaneous or intraperitoneal route, were killed when one or three hours after injection. Mice injected intravenously, were killed after ten minutes or one hour after injection. Blood, plasma and selected tissues were collected at various time points and counted in gamma counter to determine the half-period of existence in the body and tissue distribution of exogenous labeled protein. Tissues that were collected for the account, as well as the intervals of killing was chosen on the basis of reports on the distribution of other cytokines, labeled with radionuclides.

When the death of the collected tissue for counting of radioactivity included in the thymus, spleen, kidney, liver lobe, the proportion of lung and bladder. In the group receiving intraperitoneal injection, the intestine also considered to assess the frequency of occurrence of the introduction of the intestine, and in receiving subcutaneous injection of mice was considered the skin with underlying structures in the injection zone. The number of pulses per minute for a liver and a lung was calculated from the plot, which was considered, and the percentage of the weight of a body represented by this plot.

After completion of this IP is to study the collected tissue, whole blood and plasma counted on a gamma counter COBRA II AUTO-GAMMA, (Packard Instrument Company, Meriden, CT). An aliquot of the original labeled solution for injection doses also believed in the end this study with the tissues. It was possible to calculate the percentage of the total injected radioactivity for each mouse and simultaneous correction of all numbers imp./min on radioactive decay. Approximation of the rest of the blood volume and weights of the bodies showed that most of the introduced pulse was counted, and, consequently, the percentage of emulsol on the fabric were a reasonable representation of the distribution of the pulses after the introduction of the labeled zalpha11 ligand each of the paths.

C. Tagging111indium zalpha11 ligand

Purified recombinant human zalpha11 ligand person (example 29) conjugatively with a 10-fold excess of DTPA (Pierce, Rockford, II) incubation for 30 minutes at room temperature in SFR. The unreacted DTPA and the hydrolysates were removed by changing the buffer to Biomax-5k NMWL (Ultrafree-15, Millipore, Bedford, MA). Peak protein free volume was concentrated to 5 mg/ml, and take an aliquot for testing in bioanalysis (anti-CD40-stimulation of murine b-cells) (example 44)). Upon confirmation that DTPA-conjugate still has full biological activity, this conjugate was diluted to 0.5 mg/ml of 1 M sodium acetate pH 6.0. Two MCI111India absorbed in 0.5 ml of 1 M sodium acetate pH 0 and mixed with a conjugate of DTPA-zlh11-ligand person within 30 minutes at room temperature. Not included111indium was removed during the shift buffer on SFR on column PD-10 (Pharmacia, Piscataway, NJ). Radioactively labeled material was diluted unlabeled zalpha11-ligand person obtaining specific activity of 100 MCI/mg, sterile filtered and kept at 4°With during the night. One hundred percent of the labeled protein was retained on the membrane Biomax-Sk NMWL (Millipore). Labeled111indium zalpha11 ligand person was injected into mice in studies of elimination and pharmacokinetic studies. Fifty micrograms of protein zalpha11 ligand person labeled with 5 µci labeled zalpha11 ligand person, in 0.1 ml of carrier-SFR was injected in each animal.

C. a preliminary study of the distribution

After one and three hours after administration of all three paths, the highest concentrations111ln-zlph11-ligand man was found in the kidney, and the second highest concentration was in the urine and bladder, as shown in these tissues, with the highest pulse/min Average pulse/min, extracted from kidneys were 3-8 times higher than the pulse/min a liver depending on the route of injection and the point in time of the killing. For example, the average number of pulse/min kidney at 60 minutes after iv injection was 4.5 times higher than the average imp./min calculated for the whole liver from the same group. In the group that were killed in ten mine is after intravenous the highest number of pulse/min was again in the kidney, and the second highest accumulation was equivalent in the liver, the bladder and urine.

D. Preliminary pharmacokinetic study

Blood collection and plasma was carried out at 10, 30 and 60 minutes after injection in all three ways. After injection iv by separate groups of mice blood samples and plasma were taken at two, five and ten minutes. In another group of mice that received their injections ip or sp by, blood samples were taken at one, two and three hours. With regard to the processing of groups, see table 6. A short time collection cover soobshenii the period of existence in the body of IL-2 after intravenous injection. Reported T1/2 was 2.5-5.1 minutes. In relation to the introduction of in vivo IL-2, see Donohue JH and Rosenberg SA J Immunol. 130:2203, 1983. Long-term time points were chosen to determine the expected phase of elimination.

Table 6
Method injectionTime phlebotomy (minutes)Time killing
Intravenous group 12, 5, 1010 min
Intravenous group 210,30, 6060 min
Intraperitoneal group 110, 30,6060 min
Intraperitoneal g is the SCP 2 60,120, 180180 min
Subcutaneous group 110, 30, 6060 min
Subcutaneous group 260, 120, 180180 min

It was shown that IL-2 is eliminated from the serum with a half-life of existence in the body for approximately three minutes in mice after iv injection. In respect of references, see Donohue, JH and Rosenberg SA supra. After the ip and sc injection of similar amounts of IL-2 duration of the saving activity of IL-2 in serum was prolongirovanie with 2 units/ml in less than 30 minutes after iv injection to more than 2 units/ml within 2 hours after ip and 6 hours after sc injection. The main route of clearance of IL-2, apparently, are the kidneys. It was shown that zalpha11 ligand is structurally similar to IL-2, as discussed here. A preliminary assessment of the elimination of zalpha11 ligand, apparently, is consistent with the apparent clearance of IL-2 by the kidneys, on the basis of the accumulation of imp/min in kidneys, and then the bladder and urine in this study.

Were made to assess pharmacokinetic parameters based on decompartmentalise data analysis imp./min, obtained from plasma, using the RK-analysis WinNonLin, Version 1.1. (Scientific Consulting Inc., Gary, NC). Half of existence zalpha-11-ligand in plasma was estimated using the predicted rate constants of the terminology is the real elimination for intravenous, subcutaneous and intraperitoneal dose of 50 mcg. The pharmacokinetic results were evaluated due to the limited data points in the terminal region of elimination the plasma concentration versus time profiles. In addition, construction of the terminal phase of elimination for doses of sc and ip required the use of data from time points, during which it is still, apparently, is the absorbance of111ln-zalpha11 ligand person. However, estimates of half-periods of existence in the body after intravenous, subcutaneous and intraperitoneal administration of doses were 13.6 minutes, 18.8 minutes and 34.3 minutes respectively. Because the range of doses has not been evaluated, it is unclear whether there has been saturated or active elimination kinetics Michaelis-Menten). Thus, these half-periods of existence in the body are rough estimates.

Approximate estimates of the bioavailability of the labeled protein was based on the area under the curve (AUC) after subcutaneous or intraperitoneal administration of doses in comparison with the area under the curve after intravenous doses. Estimated bioavailability after subcutaneous and intraperitoneal injection was 35,8 and 63.9 per cent respectively. As explored only one dose of protein bioavailability was evaluated depending on the dose. Approximately calculated clearance and volume of distribution is possible (based on data from intravenous injection) were 0,48 and 6.1 ml/min, respectively.

Although these data are preliminary, the fate of zalpha11 ligand, administered iv, was similar fate, reported for IL-2, another cytokine with the structure of a 4-helical bundle (Donohue JH and Rosenberg SA, supra). Like IL-2 iv entered zalpha11 ligand has a half-period of existence in the plasma only minutes from the main clearance in the kidney. Three hours after injection the main part of the labeled material, australiacanada of the kidneys, are still retained by the membrane Biomax 5K NMLW (Millipore). Because earlier it was reported that India remains associated with the protein even during lysosomal degradation (Staud, F. et al., J. Pharm. Sciences 88:577-585, 1999), zalpha11 ligand accumulates and can be degraded in the kidney. The present study also showed, as was observed with many other proteins, including IL-2 (Donahue JH and Rosenberg SA, supra)that the introduction of ip and sc significantly prolonged plasma levels zalpha11 ligand.

Example 39

Selection and breeding of fresh CD34+ MNC fraction of human bone marrow using zalpha11 ligand for assessment of NK activity

A. Screening and selection of CD34+ cells from human bone marrow

Fresh mononuclear cells (MNC) of human bone marrow was obtained for enrichment in the cells having the activity of MK-cells (natural killer cells). Fresh MNC person received from Poeitic Technologies (Gaithersburg, MD). 10 ml of alpha-MEM (JRH, Lenexa, KS)containing 10% FCS HIA(Hyclone, Logan, UT) and antibiotics 1% PSN (Gibco BRL, Grand Island, NY)was added to a suspension of these cells and the cells were passed through a sieve of 100 μm. The cells are then considered, besieged, washed with 10 ml FR containing 2% FCS, then again besieged and resuspendable in 1 ml SPR containing 2% FCS. Cells that have the cell surface marker CD34 (CD34+ cells)were separated using magnetic particles using a set of Detachabead with Dynabeads M-450 CD34 (Dynal, Oslo, Norway) according to the manufacturer's instructions. As the fraction of CD34+ cells, and the fraction of CD34 - cells was further analyzed as described below.

Century Reproduction of CD34+ cells using zalpha11 ligand

The fraction of CD34+ cells were sown in four wells in 24-hole tablet. 50000 positively selected cells, suspended in 1 ml of medium alpha-MEM (JRH)containing 10% FCS HIA (Hyclone) and 1% PSN (Gibco BRL) plus various cytokines, described below, were sown in each of the 4 holes (1-4). The various reagents used to test for induced zalpha11-ligand reproduction CD34+ selected bone marrow MNC: reagents included: flt3 (R&D, Minneapolis, MN); purified zalpha11 ligand person (example 30C example 30D); IL-15 person (R&D). The reagents were combined in the following way in day 0: In well No. 1 was added 2 ng/ml human flt3. In well No. 2 was added to 2 ng/ml human flt3 and 15 ng/ml purified zalpha11 ligand person. Well No. 3 was added 2 ng/ml flt3 and 20 ng/ml IL-15 people. Well No. 4 was added 2 ng/ml human flt3, 15 ng/ml purified zalpha11 ligand person and 20 ng/ml IL-15 people. After incubation for 18 days of cell suspension from each well was besieged and then resuspendable in 0.5 ml of medium alpha MEM (JRH)containing 10% FCS HIA (Hyclone) and 1% PSN (Gibco BRL), and considered to assess the proliferation fraction of CD34+ cells. The low level of proliferation observed in the presence of only flt3 (test hole # 1), but the presence of IL-15 or zalpha11 in addition to flt3 had no significant effect on reproduction (wells # 2 and # 3). However, the reproduction of the above control flt3 was evident in hole # 4, which contained IL-15 and zalpha11 ligand, in addition to flt3. This result suggests that zalpha11 and IL-15 act synergistically in reproduction of a population of CD34+ human cells. In addition, the results of this experiment confirmed the results observed with murine zalpha11-ligand in murine BM-test (example 21).

Then all the population of cells tested for NK activity and subjected to flow cytometrical analysis, as shown below (example 41).

C. Reproduction of CD34+ or CD34 - cells using zalpha11 ligand with the delayed addition of IL-15

Both CD34+and CD34 - fractions were sown separately in wells of a 12-hole tablet (1-6). Each of the six wells contained 100000 positively or negatively selected cells in 2 ml of alpha MEM containing 10% FCS HIA and PSN, OPI is Anna above. In well No. 1 was added 2 ng/ml human flt3 in day 0. In well No. 2 was added to 2 ng/ml flt3 person per day 0 and after 5 days incubation, 20 ng/ml IL-15 people. Well No. 3 was added 2 ng/ml human flt3 and 15 ng/ml zalpha11 ligand person per day 0. Well No. 4 was added 2 ng/ml human flt3 and 15 ng/ml zalpha11 ligand person per day 0 and after 5 days incubation, 20 ng/ml IL-15 people. In well No. 5 was added 2 ng/ml human flt3 and 20 ng/ml IL-15 / person / day 0. In well No. 6 was added 2 ng/ml human flt3, 15 ng/ml zalpha11 ligand person and 20 ng/ml IL-15 at day 0. After incubation for only 15 days from the beginning of the experiment, cells from each well were collected and counted.

In the CD34+ population was observed a low level of proliferation in the presence of only flt3 (test hole # 1), but the presence of IL-15 or zalpha11 added at day 0 to flt3, had no significant effects on reproduction (wells No. 3 and No. 5). The addition of IL-15 after 5 days had a proliferative effect in comparison with flt3-control (well # 2 in comparison with well # 1) and proliferative activity in the presence of zalpha11 (well # 4 in comparison with well # 3). However, the greatest reproduction observed in hole # 6, which contained IL-15 and zalpha11 ligand in addition to flt3 in day 0.

In CD34 - populations were not observed proliferation in the presence of only flt3 (test hole # 1) and actually observed a decrease in cell population. Presence is their zalpha11, added on day 0 in addition to flt3 (well No. 3), was similar to flt3-control. The presence of IL-15, added at day 5, increased proliferating effect on the cells in the presence (cell No. 4) or in the absence (box No. 2) zalpha11 ligand. Again, the higher the reproduction was evident in hole # 6, which contained IL-15 and zalpha11 ligand, in addition to flt3, day 0.

Then all the population of cells tested on MK-activity and subjected to FACS analysis, as shown below (example 40).

Example 40

Selection and breeding of fresh murine cells using zalpha11 ligand of human and mice for assessment of NK activity and markers of NK-cells

A. Selection and breeding of fresh cells of low density bone marrow using zalpha11 ligand of human and mouse

Fresh bone marrow cells of mice were isolated by overlaying clips on both ends mouse femurs and rinse two or three milliliters of the medium for cultivation (see below) through the interior of the bone in the collection tube. The culture medium was 500 ml of medium RPMI 1640 (JRH Biosciences, Lenexa, KS), 5 ml 100x L-glutamine (Gibco BRL, Grand Island, NY); 5 ml 100x Na-pyruvate (Gibco BRL); 5 ml UK penicillin, streptomycin, neomycin (PSN) (Gibco BRL), and 50 ml V / V heat inactivated fetal calf serum (FCS) (Hyclone Laboratories, Logan, UT). Then cells in the bone marrow was destroyed by pipetting environment up and NR is C several times. These cells then besieged and washed once with culture medium and passed through a sieve of 70 μm. Then mononuclear cell low-density contributed by the exposure of bone marrow cells to a density gradient. The bone marrow cells in five to eight milliliters environment for growing gently pietravalle on the top five to eight ml NycoPrep 1,077 Animal (Nycomed, Oslo, Norway) in a centrifuge tube. Then this gradient was centrifuged at 600 × g for 20 minutes. Mononuclear cell low-density were collected from the interphase layer between NycoPrep and the environment. Then these cells were diluted to approximately 20 ml in the culture medium, besieged and washed. Then cells were sown at approximately 0,5-1,5×106cells/ml in culture medium in a standard flask for tissue culture and incubated at 37°C, 5% CO2within two hours.

Then unattached, having a low density (NA LD) bone marrow cells were collected and sown at 0.5 to 2.0×105cells per ml in culture medium plus 2.5 ng/ml murine flt3 (R&D Systems, Minneapolis, MN) plus 25-50 ng/ml interleukin 15 (IL-15) person (R&D Systems) with 50-150 ng/ml zalpha11 ligand man or without him; or from 0.12 to 10 ng/ml murine zalpha11 ligand or without it.

No significant reproduction without adding zalpha11 ligand human or mouse. Narir the captured cells were multiplied in culture, containing murine zalpha11 ligand, only at 0.12 ng/ml, and in cultures containing zalpha11 ligand man, only 22 ng/ml In cultures containing both human and mouse zalpha11 ligand, reproduction unattached cells increased with increasing dose zalpha11 ligand, and the saturating response of murine ligand is at approximately 5-10 ng/ml zalpha11 ligand person does not reach the saturating response even at the highest dose of 200 ng/ml Zalpha11 ligand man is, apparently, about 20-100 times more strong on murine cells than mouse zalpha11 ligand. After approximately five to ten days multiplied zalpha11-ligand mouse cells were collected and were analyzed by flow cytometry (FACSCalibur; Becton Dickinson, Mansfield, MA) to determine what percentage of them was positive for the antigen NK cells, and 46% were positive for the marker PanNK-DX5 cells (Pharmingen).

C. Selection and reproduction of fresh deprived line cell differentiation of mouse bone marrow

Fresh mouse deprived line differentiation (lin-) bone marrow cells were isolated from fresh cells mouse bone marrow first, incubation of these cells with the following antibodies: TER119, Gr-1, B220, MAC-1, CD3e and l-Ab (Pharmingen, San Diego, CA). Then lin+ cells were removed using sheep antibodies against rat IgG Dynabeads M-450 (Dynal, Lake Succss, NY) according to the manufacturer's instructions.

Then negatively selected lin - bone marrow cells were sown as described above, the culture medium plus either 2.5 ng/ml flt3 (R&D Systems) and 25 ng/ml IL-15 (R&D Systems), or containing flt3, IL-15 and mouse zalpha11 ligand, 2-5% KSS-mouse zalpha11 ligand air-conditioned environment. After six days of cultivation, the culture was collected, considered and provided for analysis of the activity of NK-cells (example 41). Cells grown with mouse zalpha11-ligand, were approximately two to three times more effective for lysis of target cells NK-cells (YAC-1 cells)than cells growing without zalpha11 ligand.

C. Selection and reproduction of CD4 - CD8- (double-negative or DN) thymocyte

Fresh murine thymocytes were isolated by cutting and screening Timonov from three to eight mice. Then CD4 - CD8- (DN) cells were negatively selected by incubation of thymocytes with antibodies anti-CD4 and anti-CD8 (PharMingen) then remove CD4+ CD8+ cells using sheep antibodies against rat IgG Dynabeads M-450 (Dynal) according to the manufacturer's instructions.

Then murine DN thymocytes were grown in culture medium plus 2.5 ng flt3 (R&D Systems), 25 ng/ml IL-15 (R&D Systems) and 10 ng/ml IL-7 (R&D Systems) with murine zalpha11-ligand or without it, as described above. Six days later the cells were collected, counted, were analyzed by flow cytometry as described you the e, and also provided to analyze the activity of NK-cells (example 41).

The culture that grew murine zalpha11-ligand, gave approximately 480,000 cells, whereas culture without zalpha11 ligand gave approximately 160,000 cells. It was found that the culture that grew murine zalpha11-ligand is approximately 16.2% positive antigen NK cells Pan NK, DX5 (PharMingen). The culture that grew without zalpha11 ligand was 14.6% positive DX5. Cells grown with zalpha11-ligand, literally target cells NK-cells, YAC-1, approximately two times better than cells grown without zalpha11 ligand. Multiplied cells did not literally significantly negative control line of the target cells, EL4. These results suggest that zalpha11 ligand selectively propagates lytic NK cells.

Example 41

Activity multiplied zlh11-ligand human and mouse cells and Mature murine NK cells in assays of cytotoxicity of NK-cells

A. Analysis of NK-cells

Mediated NK cell cytolysis was tested using the standard test release51Cr. Target cells (cells C ATS No. CRL-243) in the analysis of human cells and YAC-1 (ATS No. TIB-160) in the analysis of the mouse) are deprived of the molecules of the major histocompatibility complex (MHC), which gives them a mediated sensitivity to NK cell lysis. Negative control of the line the target cells in the analysis of mice is MNF +thymoma EL4 (ATSS No. TIB-39). The inventors were grown cells C, EL4 and YAC-1 among RP10 (standard RPMI 1640 (Gibco BRL, Grand Island, NY)supplemented with 10% FCS (Hyclone, Logan, UT)and 4 mm glutamine (Gibco BRL), 100 IU/ml penicillin+100 MCG/ml streptomycin (Gibco BRL), 50 μm β-mercaptoethanol (Gibco BRL) and 10 mm HEPES-buffer (Gibco BRL). On the day of analysis 1-2×106target cells were collected and resuspendable the 2.5-5×106cells/ml in medium RP10. The authors added 50-100 ál of 5 MCI/ml51Cr-sodium chromate (NEN, Boston, MA) directly to the cells and incubated them for 1 hour at 37°C, then washed them twice 12 ml SPR and resuspendable them in 2 ml of medium RP10. After counting the cells on hemocytometer target cells were diluted to 0.5-1×105cells/ml and 100 μl (0.5 to 1×104cells) were mixed with effector cells, as described below.

In the analysis of human effector cells were obtained from selected and expanded CD34+ BM cells (example 39B), which were collected, washed, counted, mixed at various concentrations with51Cr-labeled target cells in 96-well round-bottom tablets and incubated for 4 hours at 37°C. After consumerbase effector cells and labeled target cells, half of the supernatant from each well was collected and counted in a gamma counter for 1 minute per sample. The percentage of specific visualaid is of 51Cr was calculated from the formula 100 x (X-Y)/(Z-Y), where X denotes the release of51Cr in the presence of effector cells, Y denotes the spontaneous release in the absence of effectors, and Z represents the total release51Cr of target cells, incubated with 0.5% Triton X-100. The data plotted in the graph as % specific lysis depending on the relationship the effector-target in each hole.

C. Activity multiplied zalpha11-ligand human cells

Selected CD34+ LDC-human cells, cultured with flt3 +/-zalpha11 ligand and flt3 + IL-15 +/- zalpha11 ligand (example 39), collected at day 15 to assess their ability to lyse MHC-K cells in the standard analysis release51Cr, as described above, and to analyze their surface phenotype flow cytometry. As expected from previous posts, (Mrozek, E et al. Blood 87:2632-2640, 1996; Yu, H et al. Blood. 92:3647-3657, 1998), the simultaneous addition of IL-15 and flt3L really induced the growth of a small population of CD56-cells. Interestingly, although the VM cells, cultured simultaneously with zalpha11-ligand and flt3L, not multiply significantly, there was a significant increase in the total number of cells in cultures containing a combination of flt3L, zalpha11 ligand and IL-15 (see example 39).

To assess the phenotype of the surface of these VM-cultures of human authors were stained with small aliquots of these cells for cvetnogo running cytometrical analysis of mAb anti-D3-FITC, anti-D56-PE and anti-D16-CyChrome (all from PharMingen, San Diego, CA) and analyzed them on a FACSCalibur using CellQuest software (Becton Dickinson, Mountain View, CA). This flow cytometrics analysis confirmed that cells grown from these cultures were differentiated NK cells, as they were large and granular, and expressed as CD56 and CD16 were CD3-(Lanier, LL Annu. Rev. Immunol. 16:359-393, 1998). In addition, these cells showed significantly more high effector function than cells grown with IL-15 and flt3. More specifically, cells that grew in the presence of all three cytokines was literally more than 40% target C with respect to the effector: target (E:T) of 1.5, while cells growing in the presence of IL-15+flt3L, literally less than 5% of the targets at E:T, is equal to 2. These data demonstrate that, in combination with IL-15, zalpha11 ligand stimulates the differentiation of NK cells from CD34+ BM cells. C. Activity multiplied murine zalpha11-ligand cells

To test the effects of zalpha11 ligand on murine hematopoietic cells predecessors, peeled negative in relation to the line of differentiation (Lin-) bone marrow cells from mice C57B1/6 was replicated in flt3+IL-15+/- zalpha11 ligand as described in example 40B. In day 6 culture these cells ("effectors") were collected and counted, and then suspended in 0.4 ml of medium RP10 (example 41A). Two aliquots (0.15 ml each) of each sample, razmnozheny the e zalpha11-ligand or without zalpha11 ligand (example 41A), was serially diluted 3 times in duplicate in 96-well round-bottom tablets, a total of 6 wells with 100 μl each. The remaining 100 μl of the cells were stained for surface markers of NK-cells using mAb FITC-anti-V and PE-anti-OH (PharMingen) and analyzed by flow cytometry. Each group of cells subjected to the action of flt3+IL-15 in the presence or in the absence of zalpha11 ligand, had similar fractions of 2B4+DX5+ cells in 65-75% positive for both markers of NK-cells.

For analysis of NK-lysis of target cells (YAC-1 and EL4) were labeled51Cr, as described above. After the account of the target cells on hemocytometer these target cells were diluted to 0.5-1×105cells/ml and 100 ál of YAC-1 or EL4 (0.5 to 1×104cells) was mixed with 100 μl of effector cells and incubated for 4 hours at 37°C. Specific lysis was determined for each well, as described above.

The authors found that cells grown in the presence of flt3+IL-15+zalpha11 ligand, showed enhanced lytic activity (approximately 2 times) against target YAC-1 (but not killed MNF+control cell line EL4). When the ratio of effector : target (E:T) 5, NK-cells generated in the presence of all 3 cytokines (zlh11-ligand+flt3+lL-15), literally 12% of the cells YAC-1, whereas NK-cells, multiplied flt3+IL-15, literally 6% target YAC-1. Subsequent experiments confirmed this trend is the s.

In the second approach to determining the biological activity of zalpha11 ligand on murine NK cells, the authors were isolated immature CD4-CD8-(double negative, DN) mouse thymocytes as described in example 40C, and cultivated them with IL-15+flt3+IL-7 or IL-15+flt3+IL-2, with zalpha11-ligand or without zalpha11 ligand. At day 6 of culture, cells were collected and analyzed for NK lytic activity in the cells YAC-1 and EL4, as described above. The authors found that cells cultured in the presence of zalpha11 ligand, had the highest activity in this assay with enhanced lytic activity compared with cells cultured in the presence of other cytokines. Specifically, DN thymocytes grown with IL-15+flt3+IL-7, killed 18% of the cells YAC-1 at E:T 24, while cells grown in the presence of IL-15+flt3+IL-7 plus zalpha11 ligand, killed 48% of targets at the same E:So DN thymocytes grown in IL-15+flt3+IL-2, killed 15% of the target YAC-1 at E:T 6, whereas cells grown with these 3 cytokines and zalpha11-ligand, killed 35% of the cells YAC-1 at E:T 9. Flow cytometry was performed on cultured cells before analysis of NK-lysis. As was true for cultures of bone marrow, despite the proliferative action of zalpha11 ligand (number of cells was increased approximately 2 times adding zalpha11 ligand) he did not increase significantly the fraction DX5+cells (17-20% of the total number of cells in ku is Torah with IL-7 and 35-46% of the total number of cells in cultures with IL-2). These data suggest that zalpha11 ligand in combination with IL-15 and flt3 enhances the lytic activity of NK cells derived from bone marrow or thymus of mice.

R. Activity of murine zalpha11 ligand on Mature murine NK-cells

To test the action of the mouse zalpha11 ligand on Mature NK-cells, the inventors were isolated spleen of four 5-week-old mice C57B1/6 (Jackson Laboratories, Bar Harbor, ME) and crushed them between brushed on the edges of the glass object slides to obtain a cell suspension. Erythrocytes were removed by hypotonic lysis as follows: cells were besieged and the supernatant was removed by aspiration. The authors were destroyed sediment careful mixing on a vortex, then add 900 ál of sterile water with shaking, then quickly (in less than 5 seconds) were added to 100 µl of 10x HBSS (Gibco BRL). These cells then resuspendable in 10 ml of 1x HBSS and debris was removed by passing the cells through is lined with nylon mesh deformer cells (Falcon). Then these devoid of erythrocytes, the cells of the spleen was besieged and resuspendable in MACS buffer (STR+1% BSA+2 mm EDTA) and counted. The authors were stained with 300×106cells coated with anti-D5-magnetic granules (Miltenyi Biotec) and were positively selected DX5+MK-cells passing through the MACS VS+ separation column according to the manufacturer's instructions, resulting extract is of 8,4× 106DX5+cells and 251×106DX5-cells. Each of these groups of cells were cultured in 24-hole plates (0,67×106cells / well, 2 wells on one treatment option) in the environment RP10 (example 41A) alone or with 1) 30 ng/ml murine zalpha11 ligand, 2) 30 ng/ml recombinant murine IL-2 (R&D Systems, Inc., Minneapolis, MN), 3) 30 ng/ml recombinant IL-15 person (R&D), 4) 30 ng/ml each of mouse zalpha11 ligand and IL-15 human, or 5) 30 ng/ml each of mlL-2 and hlL-15. Cells were collected after 21 hours, washed and resuspendable environment RP10 and considered. The cells are then analyzed for their ability to lyse51Cr-labeled target cells YAC-1 and EL4, as described in example 41A.

Usually was low NK activity of DX5-(not NK-ketki) groups, but DX5-cells cultured with zalpha11-ligand and hlL-15, was really literally 25% of the target cells YAC-1 at E:T 82. For comparison, DX5-cells cultured only with hlL-15, literally 14% of the target cells YAC-1 at E:T 110. This suggests that zalpha11 ligand and IL-15 are working together on the remaining NK1.1+NK-cells in this preparation cells. As for drugs DX5-cells, processing one mouse zalpha11-ligand did not increase significantly their effector function (lysis of cells YAC-1 was similar to the lysis of untreated group). As expected, both IL-2 and IL-15 significantly improved NK is aktivnosti. However, the highest level of lysis was detected in the group treated zalphall-ligand and hlL-15 (65% lysis of cells YAC-1 at E:T of 3.3, compared to 45% lysis at E:T 4 for group processing hlL-15). Taken together, these results suggest that although one zalpha11 ligand may not increase NK cell lytic activity, it really enhances NK lytic activity of Mature NK cells in the introduction to IL-15.

Example 42

Proliferation under the action of zalpha11 ligand of the human and murine T cells in the proliferation test of the T-cells

A. the Proliferation of murine zalpha11-ligand murine T cells

T cells from mice C57B1/6 (Jackson Laboratories, Bar Harbor, ME) were isolated from the United splenocytes and lymphocytes from the axillary, brachial, inguinal, cervical, and mesenteric lymph nodes (LN). Spleen kneaded brushed on the edges of the glass object slides to obtain a cell suspension. LN (lymph nodes) tore it apart with tweezers and was passed through the deformer cells to remove debris. Pooled splenocytes and LN cells were divided into subpopulations of CD8+and CD4+using two consecutive MACS magnetic separation column according to the manufacturer's instructions (Miltenyi Biotec, Auburn, CA). The whole thymocytes were collected from the same mice.

Lekti were cultured with 3×105cells per well (Timo is the ITA) or 10 5cells per well (Mature T cells) with increasing concentrations of purified mouse zalphall-ligand (0-30 ng/ml) (example 24 and example 29) in 96-well flat-bottomed tablets, pre-coated overnight at 4°With various concentrations of anti-D3 mAb 2C11 (PharMingen)for 3 days at 37°C. the Antibody anti-D30 served for activation of murine T cells through T-cell receptor. To each well was added 1 µci3H-thymidine on day 2 and the tablets were collected and counted after 16 hours to assess cell proliferation.

When tested zalpha11 ligand in the test proliferation of T cells, the authors found that he would costimulatory anti-D3-activated murine thymocytes, leading to accelerated growth CD8+CD4+cells (most thymocytes cultured with anti-D3+zlh11-ligand, were CD8+CD4+on day 3 of culture, while cells cultured only with anti-D3 not inclined significantly to this phenotype to day 5). The authors did not observe significant levels of proliferation of thymocytes in response to zalpha11 ligand in the absence of anti-D3.

Interestingly, when the authors analyzed the Mature murine T cells for their ability to respond to zlh11-ligand+anti-D3, they found that only CD8+but not CD4+the subpopulation, replied dependent on dose on zalpha11 ligand. The authors also observed a weak, but reproducible, the cast new is ERATIO CD8 +cells (but not CD4-cells) in response to one zalpha11 ligand. Interestingly, this was not observed for T cells (see example 42, below).

Century Proliferation zalpha11-ligand human T-cells

CD4+and CD8+T-cells were isolated from RPMS as described in example 43 (below). Cells were cultured at approximately 105cells per well with increasing concentrations of purified zalpha11 ligand person (0-50 ng/ml) (example 24) in 96-well flat-bottomed tablets, pre-coated overnight at 4°With various concentrations of anti-D3 mAb UCHT1 (PharMingen)for 3 days at 37°C. To each well was added 1 µci3H-thymidine on day 2 and the tablets were collected and counted after 16 hours to assess cell proliferation. In contrast to the results obtained by the authors with murine T-cells, preliminary data suggest that zalpha11 ligand person will costimulated CD4+but not CD8+, T cells of a person dependent on dose.

Example 43

Poland real-time shows the expression of zalpha11 ligand in CD4+ cells

A. Purified T cells as the primary source used to assess the expression of zalpha11 ligand person

Whole blood (150 ml) was taken from healthy human donors and mixed 1:1 with SFR in conical tubes 50 ml Then thirty ml of the diluted blood was put on the 15 is l ficoll Ficoll Paque Plus (Amersham Pharmada Biotech, Uppsala, Sweden). These gradients were centrifuged for 30 minutes at 500 g and gave the centrifuge will stop without braking. Devoid of red blood cells cells in interphase (RVMS) were collected and washed 3 times FR. Output selected mononuclear cells in peripheral blood (RVMS) was 200×106before selection, described below.

RVMS suspended in 1.5 ml of MACS buffer (FR, 0.5% EDTA, 2 mm EDTA) and 3×106cells were separated for the control RNA and running cytometrical analysis. The authors then added 0.25 ml of microgranules with antibody anti-human CD8 (Miltenyi Biotec) and the mixture is incubated for 15 minutes at 4°C. These cells labeled with CD8-granules, washed with 30 ml of MACS buffer and then resuspendable in 2 ml of MACS buffer. VS+column (Miltenyi) were prepared in accordance with the manufacturer's instructions. Then VS+the column was placed in a magnetic field Vario MACS (Miltenyi). The column was balanced with 5 ml of MACS buffer. Then the selected primary mouse cells were applied on the column. CD8-negative cells were made to pass through the column. The column is washed with 9 ml (3×3 ml MACS buffer. Then the column was removed from the magnet and placed in a falcon tube 15 ml CD8+ cells were suirable by adding 5 ml of MASC-buffer to the column and the bound cells were washed using a piston (plunger)provided by the manufacturer. The total yield of CD8+ selected peripheral cells the cells was 51× 106cells. CD8-negative flowing through the column, the cells were collected, counted, stained with antibody-coated anti-CD4 granules, then incubated and passed through the new VS+ column at the same concentration as described above. The total yield of CD4+ - selected peripheral T cells was 42×106cells.

A sample of each of the CD8+ and CD4+ selected T-cells was removed for staining and sorting cell sorting device with activation of fluorescence (FACS) to assess their purity. PE-conjugated antibody against CD4 human antibody against CD8-FITC Ab and antibody against CD19-CyChrome Ab (all from PharMingen) were used for staining of CD8+ and CD4+ selected cells. CD8+ selected cells in the first experiment were 80% CD8+ cells, and CD4+ selected cells were 85% CD4+ cells. In two subsequent experiments (example V) CD8+ purified cells had a purity of 84% and 81%, and CD4+ cells had a purity of 85% and 97%, respectively. In one experiment, the authors were stained nesviazana (passing through the column) cells covered aHTH-human CD19 granules (Miltenyi) and ran them through a third column with magnetic granules to highlight CD19+ b-cells (they had a purity of 92%).

CD8+, CD4+ and CD19+ selected cells were activated by incubation with 0.5×106cells/ml in RPMI + 5% ultramylonite man (Gemini Bioproducts, Calabasas, CA) + PMA 10 ng/ml and Ion the Qing 0.5 μg/ml (Calbiochem) for approximately 4, 16 or 24 hours at 37°C. T cells (2,5×106per well) additionally stimulated in 24-hole tablets, pre-coated overnight with 0.5 μg/ml tablet-linked mAb anti-CD3 UCHT1 (PharMingen) with soluble mAb anti-D28 (PharMingen) at 5 µg/ml or without them. At each time point, cells were collected, besieged, washed once SFR and again besieged. The supernatant was removed and the precipitates were immediately frozen in a bath with a mixture of dry ice/ethanol, and then stored at -80°to obtain in the subsequent RNA.

Real-time PCR was performed on these CD8+, CD4+ and CD19+ selected cells as described in example V and 43C and below, to evaluate the expression of zalpha11 ligand and zalpha11 receptor human.

C. Primers and probes for quantitative RT-PCR for the expression of zalpha11 ligand person

Quantitative RT-PCR real-time use of the system for detection of sequences ABI PRISM 7700 (PE Applied Biosystems, Inc., Foster City, CA) was described previously (See. Heid, C.A. et al., Genome Research 6:986-994, 1996; Gibson, U.E.M. et al., Genome Research 6:995-1001, 1996; and Sundaresan, S. et al., Endocrinology 139:4756-4764, 1998. This method involves applying a gene-specific probe containing as a reporter, and a quenching dye. When the probe is intact, the radiation of the reporter dye is extinguished because of the close proximity to the quenching dye. During PCR extension using gene-specific the x forward and reverse primers, the probe is cleaved by 5'-nuclease activity of the Taq polymerase, that releases the reporter dye from the probe, resulting in an increase in fluorescent emission.

The primers and probes used for quantitative RT-PCR analysis real-time constructed using software for designing primers Primer Express™ (PE Applied Biosystems). The primers for zalpha11 ligand person constructed extending from the grasp of the intron-exon junction to eliminate amplification of genomic DNA. Direct primer, ZC22281 (SEQ ID NO:90), and reverse primer, ZC22279 (SEQ ID NO:91)was used at a concentration of approximately 300 nm for the synthesis of the product 80 BP Corresponding probe zalpha11 ligand TaqMan, ZG32 (SEQ ID NO:92) was synthesized and labeled PE Applied Biosystems. This probe was labelled at the 5'end of the reporter fluorescent dye (6-carboxyfluorescein) (FAM) (PE Applied Biosystems) and at the 3'-end of the quenching fluorescent dye (6-carboxytetramethyl) (TAMRA) (PE Applied Biosystems). For testing integrity or quality of all RNA samples were subjected to screening for rRNA using a set of primers and probes ordered from PE Applied Biosystems (cat. No. 4304483). Reporter fluorescent dye for this probe is VIC (PE Applied Biosystems). Results for rRNA will allow you to normalize the results for zalpha11-ligands.

RNA was obtained from precipitation, obtained in example 43A, using RNeasy kit Miiprep™ (Qiagen, Valencia, CA) according to the manufacturer's instructions. Control RNA was obtained from approximately 10 million KSS-expressing cells zlh11-ligand person.

C. Primers and probes for quantitative RT-PCR for the expression of zalpha11 receptor human

Real-time PCR was performed to assess the expression of zalpha11 receptor as in example V and example 43D, using cells obtained under the conditions described in 43A, and probes specific for the zalpha11 receptor. Direct primer, ZC22277 (SEQ ID NO:93), and reverse primer, ZC22276 (SEQ ID NO:94), were used in PCR reactions (above) at a concentration of approximately 300 nm for the synthesis product 143 BP Corresponding probe zalpha11 TaqMan®named ZG31 (SEQ ID NO:95) was synthesized and labeled PE Applied Biosystems. RNA from BaF3 cells expressing zalpha11 receptor human, used to generate the appropriate control for the standard curves for real-time PCR as described in example 43D below.

D. Quantitative RT-PCR real-time

Relative RNA levels zalpha11 ligand was determined by analysis of samples of total RNA using the one-stage method RT-PCR (PE Applied Biosystems). RNA from cells KSS expressing zalpha11 ligand man, was isolated by standard methods and used to obtain the standard curve. The curve consisted of serial dilution in the range of the 2,5-2,5× 10-4ng for screening rRNA and 25-0,0025 ng for screening zalpha11 ligand, and each point of the standard curve were analyzed in three replicates. Samples of total RNA was also analyzed in triplicate in the levels of transcript zalpha11 ligand person and on the levels of rRNA as endogenous control. Each one-step RT-PCR reaction contained 25 ng of total RNA in buffer A (50 mm KCl, 10 mm Tris-HCl); internal standard dye (ROX) PE Applied Biosystems)); suitable primers (50 nm for rRNA samples, 300 nm for samples zalpha11 ligand) and probe (50 nm for rRNA, 100 nm for zalpha11 ligand), 5.5 mm MgCl2; 300 μm each d-CTP, d-ATP, and d-GTP and 600 μm d-UTP; reverse transcriptase (0.25 u/ml); DNA polymerase AmpliTaq (Of 0.025 U/μl) and RNase inhibitor (0.4 u/μl) in a total volume of 25 µl. thermal Cycling conditions PCR were as follows: initial stage of reverse transcription (FROM) at 48°C for 30 minutes; stage activation of AmpliTaq Gold at 95°C for 10 minutes; followed by 40 cycles of amplification at 95°C for 15 seconds and 60°C for 1 minute. Relative RNA levels zalpha11 ligand was determined using standard curves as described in User Bulletin No. 2 (PE Biosystems; User Bulletin #2: ABI Prism 7700 Sequence Detection System, Relative Quantitation of Gene Expression, December 11, 1997), using measurements rRNA to normalize levels zalpha11 ligand. The samples were compared relative to calibrate the RA in each experiment. The calibrator was arbitrarily chosen on the basis of RNA of good quality and the level of expression, with which other samples could be subjected to a meaningful comparison. The results of these experiments, analyzing the expression of zalpha11 ligand and zalpha11 receptor in stimulated and unstimulated cells (example 43A), described in example 43TH below.

E. Expression of zalpha11 receptor and zalpha11 ligand person in CD4+, CD8+ and CD19+ cells

The first experiment used RT-PCR described above, to evaluate the expression of zalpha11 receptor in unstimulated and stimulated anti-D3 CD4+ and CD8+ samples at time points 0 h (estimulando ("resting" cells) and at 4 h, 15,5 h and 24 h after stimulation. That rests CD4+ sample was arbitrarily chosen as the calibrator, and imputed value of 1.00. Observed approximately 4-fold increase in the expression of the receptor in unstimulated CD4+ cells from 4 h to 24 h of cultivation and approximately 8-fold increase over the same period of time in anti-CD3-stimulated CD4+ cells. CD8+ cells found a 7-fold increase in the expression of zalpha11 receptor, with a peak at 4 h and decrease over time. When anti-CD3-stimulation of CD8+ cells had a constant 8-fold increase in the expression of the receptor.

This first experiment used RT-PCR to assess the expression of zalpha11 ligand is in the same anti-CD3-stimulated and unstimulated CD4+ and CD8+ samples. 4 h anti-CD3-stimulated CD8+ sample was arbitrarily chosen as the calibrator and the imputed value of 1.00. The results showed that estimulando CD4+ and CD8+ cells are not expressed zalpha11 ligand. The authors observed a significant increase in the expression of anti-CD3-stimulated CD4+ cells at 4 h and approximately 300-fold increase in signal observed at 15,5 hours CD8+ cells expressed a small amount of the ligand with anti-D3-stimulation, but it may have been caused by contamination of the CD8+ population by a small number of CD4+ cells.

The second experiment used RT-PCR to assess the expression of zalpha11 receptor in anti-D3-stimulated, PMA + Ionomycin-stimulated and unstimulated CD4+ and CD8+ samples at time points 0 h, and at 3.5 h, 16 h and 24 h after activation. That rests CD8+ sample was arbitrarily chosen as the calibrator and the imputed value of 1.00. Resting CD4+ and CD8+ cells are not found in significant quantities in the expression of the receptor. Expression was approximately 3 times higher in PMA + Ionomycin-stimulated CD4+ samples at 3.5 h, 16 h and 24 h after stimulation. Expression in anti-CD3-activated CD4+ cells had a maximum of 10 times above background levels at 3.5 h after stimulation, then it decreased to levels 4 times higher than the background, at 16 h after stimulation. CD8+ cells found 4 beautiful, the increase in expression at 3.5 h after PMA + Ionomycin-stimulation, moreover, the expression was decreased in subsequent time points. As in the first experiment, anti-CD3-stimulated CD8+ cells again showed 8-fold induction above background expression of the receptor.

These samples from the second experiment were also used to assess the expression of zalpha11 ligand. 24 h stimulirovannuyu PMA and Ionomycin CD4+ sample was arbitrarily chosen as a calibrator, and she was assigned a value of 1.00. The results showed that estimulando cells are not expressed zalpha11 ligand. Watched about 30-fold induction of expression of the ligand in CD4+ cells stimulated with antibody anti-CD3, 3.5 h, as seen in the previous experiment (4 h). However, there was only approximately 5-fold induction when PMA + Ionomycin-stimulation at 3.5 h, which decreased at subsequent time points. Again CD8+ cells expressed a very small amount of ligand that was possibly associated with pollution of CD4+ cells.

The last experiment used RT-PCR to assess the expression of zalpha11 receptor in anti-CD3 - and anti-D3/anti-D28-stimulated and unstimulated CD4+ and CD8+ samples at time points 0 h, 2 h, 4 h and 16 h after stimulation. CD19+ cells, activated, PMA + Ionomycin, were also subjected to screening for receptor expression at the same time intervals. That rests CD4+ sample was pressed is Ino chosen as the calibrator, and she was assigned a value of 1.00. 2 h anti-D3-stimulirovannye CD4+ cells had only a 4-fold induction of the receptor compared with 10-fold induction observed at 3.5 h in the previous experiment. The combination of anti-CD3 and anti-CD28 increased expression of zalpha11 receptor to 8-fold in comparison with the background. 16 h with anti-CD3/anti-CD28-stimulated CD4+ cells had very low levels of zalpha11 receptor as seen in CD8+ cells in previous experiments (see above). CD19+ cells, stimulated PMA + Ionomycin, was the most significant expression of zalpha11 receptor with a 19-fold increase at 2 h, but the expression levels declined again to levels of resting cells at 16 hours.

These samples from the last experiment was also used to assess zalpha11 ligand using RT-PCR. 16 h with anti-CD3/anti-CD28-stimulated CD8+ sample was randomly selected as a calibrator, and she was assigned a value of 1.00. These results showed that at 2 h of CD4+ cells was approximately 2-fold induction of expression zalphall-ligand with anti-CD3-stimulation and 5-fold induction with anti-CD3 plus anti-CD28 stimulation. These conditions of stimulation induced the expression of the ligand in time with 16 h, stimulated CD4+ cells, showing expression levels 70 times higher in comparison with the background. CD8+ and CD19+ cells did not detect the expression of zalpha11 ligand.

A certain degree of variation expected the camping between takes blood (i.e. multiple samples at different time points from the same patient and between multiple patients). Thus, trend data were analyzed in each study or from individual blood samples, and three of the above experiment were compared for overall conclusions. The trend obtained from experiments real-time PCR, is that of all the tested types of cells, CD19+ b cells, activated, PMA + Ionomycin, expressed higher levels of RNA zalpha11 receptor. CD4+ and CD8+ cells can be induced for expression of the receptor, but at lower levels than observed in b cells. Zalpha11 ligand expressively almost exclusively in stimulated CD4+ T-cells (but not in CD8+ T cells or CD19+ b cells). Although stimulation of PMA + Ionomycin induced a good signal zalpha11 ligand in this test, a significantly higher signal received from CD4+ T cells stimulated mAb anti-CD3, or a combination of mAb anti-CD3 and anti-CD28, i.e. under conditions that better mimic the clash of antigens in vivo.

Example 44

Zalpha11 ligand-dependent proliferation of b cells stimulated by antibodies, anti-CD40 or anti-lgM

A. Purification of human cells

The vial containing 1×108frozen subjected to apheresis mononuclear cells of peripheral blood (the Navy), quickly thawed in a water bath at 37°and resuspendable in 25 ml of medium for b-cells (RPMI-1640 medium (JRH Biosciences, Lenexa, KS),10% inactivated by heating fetal calf serum, 5% L-glutamine, 5% pen/strap) (Gibco BRL) in a test tube 50 ml (Falcon, VWR, Seattle, WA). The cells were tested for viability using Trypanosoma blue (GIBCO BRL). Ten milliliters of a mixture of Ficoll/Hypaque Plus (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) was layered under a suspension of the cells and was turned off for 30 minutes at 1800 rpm and allowed to stay off the brake. Then the interfacial layer was removed and transferred into a fresh Falcon tube 50 ml, brought to a final volume of 40 ml FR and off for 10 minutes at 1200 rpm with included braking. The viability of the selected b-cells was tested with Trypanosoma blue. Alternatively, freshly isolated from human blood was diluted 1:1 SFR (Gibco BRL) and were layered on Ficoll/Hypaque Plus (Pharmacia), Unscrew and washed as described above. Cells isolated from fresh or frozen source, gave equivalent results.

B cells were purified from floating on ficoll blood cells of healthy human donors (see above) using coated with anti-D19 magnetic granules (Miitenyi Biotec, Auburn, CA) according to the manufacturer's instructions. The purity of the obtained preparations were subjected to monitoring by using protocoloptions analysis with antibodies anti-D22 FITC (Pharmingen, San Diego, CA). Usually drugs In cells had a purity of >90%.

C. Purification of murine b-cells

A suspension of murine splenocytes were obtained by tearing spleens of adult mice C57B1/6 (Charles River Laboratories, Wilmington, MA) bent needles in the environment for b-cells. Erythrocytes were removed by hypotonic lysis. CD43-positive cells were removed D43-magnetic granules (Miitenyi Biotec) according to the manufacturer's instructions. The purity of the obtained preparations were subjected to monitoring using flow cytometrical analysis with antibodies anti-D45 FITC Ab (Pharmingen). Usually drugs In cells had a purity of >90%.

C. the Proliferation of anti-CD40-stimulated b-cells in the presence of zalpha11 ligand human or mouse

In cells from human or murine source resuspendable at a final concentration of 1×106cells in the medium for b-cells and were sown at 100 μl per well in 96-well U-bottom tablet (Falcon, VWR)containing various conditions of stimulation, leading to a final volume of 200 µl per well. For anti-CD40-stimulated cultures cell culture was supplemented with 1 μg/ml anti-human CD40 (Genzyme, Cambridge, MA), and mouse culture was supplemented with 1 μg/ml anti-mouse CD40 (Serotec, UK). Zalpha11 ligand human or mouse was added at dilutions ranging from 1 PG/ml to 100 ng/ml Specificity of action of zalpha11 ligand was confirmed by the inhibition of zalpha11 ligand 25 mg/ml dissolve is image receptor zalpha11CEE person (example 10A). All processing was performed in three replications. Then these cells were incubated at 37°With humid thermostat to 120 hours (man) or 72 hours (mouse). For sixteen hours before collecting all wells were added 1 µci3H-thymidine (Amersham, Piscataway, NJ) to assess whether there has been a proliferation of b-cells. Cells were collected in 96-well filter tablet (UniFilter GF/C, Packard, Meriden, CT) using a harvester cells (Packard) and collected in accordance with the manufacturer's instructions. The tablets were dried at 55°C for 20-30 minutes and the bottom of the hole was closed up opaque sealant for tablets. To each well was added 0.25 ml of scintillation fluid (Microscint-O, Packard) and the plate was read using a scintillation counter for microplate TopCount (Packard).

Incubation zalpha11 ligand at concentrations of 3 ng/ml or more increased proliferation induced by soluble anti-CD40-dependent dose as in murine b cells and In human cells in 30 times. Mouse and human b cells responded equally well as their corresponding zalpha11 ligand. In both cases, the stimulation was specific in relation to zalpha11 ligand, as it turned (shot) by the presence of soluble zalpha11 receptor in culture.

D the Proliferation of anti-lgM-stimulated b-cells in the presence of zalpha11 ligand brow the ESA or mouse

In cells from human or murine source, as described above (example 44A and example V), were sown as described above (example C). For anti-lgM-stimulation of human cells tablets pre-coated overnight with 10 mg/ml F(ab')2, lgM antibodies against human (Southern Biotech Assiciates, Birmingham, Alabama) and washed with a sterile environment prior to use. The culture was supplemented with 0-10 ng/ml hu rlL-4 (R&D Systems, Minneapolis, MN). For anti-lgM stimulation of murine cells soluble antibody anti-lgM (Biosource, Camarillo, CA) was added to the cultures at 10 mg/ml To each of the preceding conditions anti-lgM/L-4 was added zalpha11 ligand human or mouse at dilutions from 1 PG/ml to 100 ng/ml, as described above. The specificity of action of zalpha11 ligand was confirmed by inhibition of soluble zalpha11 receptor human, as described above (example C). All processing was performed in three replications. Then these cells were incubated, marked3H-thymidine, were collected and analyzed as described in example S.

Incubation with zalpha11-ligand at concentrations of 0.3 ng/ml or more inhibited proliferation, induced insoluble antibody anti-lgM (mouse) or anti-lgM and IL-4 (human) dependent on dose. This inhibition was specific in relation to zalpha11 ligand, as it is addressed by the presence of soluble zalpha11 receptor in culture.

When is EP 45

Expression of soluble zalpha11 receptor of human rights in E. coli

A. Designing expressing vector PCZR225, which expresses fused polypeptide huzalphal 1/MBP-6H

The expression plasmid containing polynucleotide encoding a soluble zalpha11 receptor human, fused at the C-end with malesurvivor protein (ICBMs), designed by homologous recombination. Fragment zalpha11 cDNA of human (SEQ ID NO:7) was isolated using PCR. The polynucleotide sequence for the fused polypeptide MBP-soluble zalpha11 receptor shown in SEQ ID NO:96. Two primers were used in obtaining zalpha11 fragment of a man in a PCR reaction: (1) Primer ZC20187 (SEQ ID NO:98), containing 40 BP of the vector flanking sequence and 25 BP corresponding to the amino-end of the zalpha11 person, and (2) primer ZC20185 (SEQ ID NO:99), containing 40 BP of the 3'- end corresponding to the flanking vector sequence and 25 BP corresponding to carboxyl-end of the zalpha11 person. Conditions for PCR reactions were as follows: 25 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 1 minute, followed by soaking at 4°C, the reaction was carried out in two replications. Two μl of the 100 μl PCR reaction was subjected to electrophoresis on a 1.0% agarose gel with 1 x TBE-buffer for analysis and observed the expected fragment of approximately 660 BP Remaining 90 μl of P Is R-reaction was combined with the second PCR tube, besieged 400 μl of absolute ethanol. Precipitated DNA was used for recombination in cut Serial recipient vector char (example 31) to obtain the design, coding fused MBP-zalpha11. Clones transformed, identified and cultivated as described in example 31. Positive clones were named pCZR225 and subjected to sequencing analysis. The polynucleotide sequence for MBP-soluble zalpha11 receptor shown in SEQ ID NO:96, and the corresponding polypeptide sequence is shown in SEQ ID NO:97. Positive clones were used for cultivation in E. coli as described in example 34, to clear fused protein huzalpha11/MBP-6H (example 46 below).

Example 46

Purification of fused protein of soluble receptor huzalpha11/MBP-6H from the fermentation of E. coli

Unless otherwise noted, all operations were carried out at 4°C. the Following procedure was used for the purification of fused protein of soluble receptor polypeptide huzalpha11/MBP-6H. Cells of E. coli containing the design pCZR225 and expressing soluble receptor huzalpha11/MBP-6H (example 45), were grown in SuperBroth II (12 g/l casein, 24 g/l yeast extract, 11.4 g/l dicale-phosphate, 1.7 g/l monopotassium phosphate; Becton Dickinson, Cockeysville, MD) and were frozen in 0.5% glycerol. Twenty grams of frozen cells in SuperBroth II + glycerin used for protein purification. Frozen cells were thawed and diluted 1:10 vrestore proteasome inhibitor (buffer for extraction) before lysis of these cells and release soluble receptor protein huzalpha11/MBP-6H. Diluted cells contained a final concentration of 20 mm Tris (JT Baker, Philipsburg, NJ), 100 mm sodium chloride (NaCl, Mallinkrodt, Paris, KY), 0,5 mm phenylmethylsulfonyl (RMSF, Sigma Chemical Co., St. Louis, MO), 2 mg/ml leupeptin (Fluka, Switzerland) and 2 μg/ml Aprotinin (Sigma). System cell disruption French Press (Constant Systems Ltd., Warwick, UK) with temperatures ranging from -7 to -10°and 30 EC per square inch was used for lysis of the cells. Diluted cells were tested to destruction readings And600before and after French Press. Lysed cells were centrifuged at 18000 g for 45 minutes to remove the debris of the destroyed cells and the supernatant was used for protein purification. The total protein concentration of the supernatant was determined by BSA-method (Pierce, Rockford, IL) according to the manufacturer's instructions.

The substrate column (25 ml) Talon metal Affinity resin (Clontech, Palo Alto, CA) (prepared as described below) was poured in a glass column, Bio-Rad, 2.5 cm (diameter) × 10 cm (height). The column was Packed and balanced under the action of gravity 10 column volumes (KO) buffer to balance Talon (20 mm Tris, 100 mm NaCl, pH 8.0). The supernatant was periodically applied to the affinity resin Talon metal Affinity resin and rocked throughout the night. The resin was poured back into the column and washed with 10 column volumes of buffer to balance Talon under the action of gravity, and then elution was performed under the action of gravity 140 ml of buffer DL is slutie (buffer to balance Talon + 200 mm imidazole, Fluka Chemical). Talon-column purified 5 column volumes of 20 mm 2-(N-morpholino)econsultancy acid pH 5.0 (MES, Sigma), 5 column volumes of distilled H2O, and then kept in a mixture of 20% ethanol/0.1% of sodium azide. Faction fourteen ml were collected throughout elsinoe chromatography and fractions were read by absorption at 280 and 320 nm and the protein was determined by BSA-way; passing through the column, and wash pools well maintained and analyzed. Fractions of elution protein of interest were pooled and applied directly on Aminosol resin (New England Biolabs, Beverly, MA).

For more pure polypeptide huzalpha11/MBP-6H combined fractions elution with Talon-affinity column was subjected to chromatography on Amylose resin (22 ml) at pH 7.4. A column of Bio-Rad with a diameter of 2.5 cm and a height of 10 cm was filled, Packed and balanced in 10 column volumes Amelonado buffer for equilibration (20 mm Tris (JT Baker), 100 mm NaCl (Mallinkrodt), 1 mm PMSF (Sigma), 10 mm beta-mercaptoethanol (TOGETHER, ICN Biomedicals Inc., Aurora, OH) pH 7.4. The sample was applied under the action of gravity at the speed of a current of 0.5 ml/min. and the Column washed with 10 column volumes Amelonado buffer for equilibration, then suirable approximately 2 column volumes of a mixture Amelody buffer to balance +10 mm maltose (Fluka, Biochemical Switzerland) under the action of gravity. Fractions of 5 ml were collected at protag the Institute all chromatography and measured the absorbance at 280 and 320 nm. Aminosol column regenerates 1 column volume of distilled H2O, 5 column volumes of 0.1% (mass/about) LTOs (Sigma), 5 column volumes of distilled H2And then 5 column volumes Amelonado buffer for equilibration.

Interest fractions were combined and were dialyzed in Slide-A - Lyzer (Pierce) with 4 × 4L SFR pH 7.4 (Sigma) to remove low molecular weight impurities, change buffer and desalted. After shifts SFR the collected material was a purified polypeptide huzalpha11/MBP-6H. The purified polypeptide huzalpha11/MBP-6H were analyzed by electrophoresis on LTO-page with staining of Kumasi and Western blot analysis with anti-rabbit antibody conjugated with horseradish peroxidase (HRP) (Rockland, Gilbertsville, PA). The concentration of the polypeptide huzalpha11/MBP-6H was 1,92 mg/ml, as defined by the BSA-analysis.

The purified polypeptide huzalpha11/MBP-6H was prepared for injection into rabbits and sent to R & R Research and Development (Stanwood, WA) to generate antibodies. Rabbits were injected with to obtain serum against huzalpha11/MBP-6H (example 47 below).

Example 47

Polyclonal antibodies to zalpha11-receptor

Polyclonal antibodies were obtained by immunization of two female new Zealand white rabbits with purified polypeptide huzalpha11/MBP-6H (example 46) or purified recombinant soluble receptor zalpha11CEE (example 10A). The corresponding polyclonal anti who ate were named rabbit antibody anti-huzalph11/MBP-6N and rabbit antibody anti-huzalpha11CEE-BHK, respectively. Each rabbit received an initial intraperitoneal injection (IP) of 200 mg of purified protein in complete Freund's adjuvant (Pierce, Rockford, IL) with subsequent booster IP injections of 100 mg of purified protein in incomplete Freund's adjuvant every three weeks. After seven to ten weeks after the introduction of the third booster injection was made krovoisliania animals and collect the serum. Then the rabbits continued to enter the booster injection and took blood every three weeks.

Zalpha11-specific polyclonal antibodies were affinity purified from sera of rabbits using column CNBr-SEPHAROSE 4B protein (Pharmada LKB)that was prepared using 10 mg of purified polypeptide huzalpha11/MBP-6H (example 32) per gram CNBr-SEPHAROSE, followed 20X dialysis in TFR during the night. Zalpha11-specific antibodies were characterized by scanning titer using ELISA using 1 mg/ml of the appropriate protein antigen as a target antibody. The lower threshold of detection (LLD) of the rabbit affinity purified antibody anti-huzalpha11/MBP-6H is the breeding of 500 PG/ml LLD rabbit affinity purified antibody anti-huzalpha11-CEE-BHK is breeding 50 PG/ml

Example 48

The distribution of zalpha11 receptor

To evaluate the distribution of zalpha11 receptor on various cell types, the authors of the invention have been rabbit polyclonal and mouse monoclonal antibodies (mAb), the direction of the Lenna against the receptor of the person (example 35 and example 47) and conjugatively these antibodies with Biotin for use in flow cytometry. First, the authors used a polyclonal antibody, which had a relatively low affinity for staining panel of cell lines: cells BaF3 wild-type IL-3-dependent murine line b-progenitor cells (Palacios and Steinmetz, ibid.4 Mathey-Prevot et al., ibid.); the BaF3 cells, transfetsirovannyh human zalpha11 (example 4); line cell lymphoma, Burkitt's lymphoma Raji (ATCC No. CCL-86), Ramos (ATCC No. CRL-1596), RPMI 8226 (ATCC No. CCL-155) and Daudi (ATCC No. CCL-213); cell line Jurkat T-cell leukemia human (ATCC No. TIB-152); cell line Thr-1 myelomonocytic leukemia person (ATCC No. TIB-202) and UT937 (ATCC No. CRL-1593.2); Pro-myelomonocytic cells HLT60 person (ATCC No. CCL-240); cell line A20 murine b-cell lymphoma (ATCC No. TIB-208); and the cell line EL4 mouse thymoma (ATCC No. TIB-39).

These cells were collected, washed once proryvnym FACS buffer with serum (WBS). WBS consisted of balanced salt Hanks solution (Gibco BRL) + 10 mm HEPES (Gibco BRL) + 1% BSA (Sigma) + 10% normal goat serum (Gemini Bioproducts, Woodland, CA) + 10% normal rabbit serum (Sigma). The washing buffer (WB) was identical WBS except that he was serum-free. After washing the cells resuspendable in 100ál WB, containing 10 μg/ml rabbit polyclonal antibodies against zalpha11 (example 47). Cells kept on ice with the antibody for 20 minutes, then washed WB and resuspendable in WB, containing goat anti-rabbit-FITC antibody (BioSource International), a incubated for another 20 minutes on ice, then washed and resuspendable 400 ál WB for analysis on a flow cytometer FACSCalibur (Becton Dickinson). Control samples were stained with only the secondary goat anti-rabbit-FITC antibody. Positive staining was defined as an offset above coloring only the secondary antibody. Although polyclonal antibodies were antibodies of low affinity, the authors confidently (significant) were detected expression of zalpha11 on transfectant BaF3/zalpha11 on all four lymphoma Burkitt person (Raji, Ramos, Daudi and RPMI 8226) and T-cells Jurkat. Resting (undifferentiated) cells HL-60 was not linked antibody anti-zalphal 1, but the authors found no positive signal on the cells HL-60 activated for 20 hours PMA (Calbiochem, La Jolla, CA), which induces differentiation of cells HL-60 monocytic-like cell. The authors also observed a positive signal on the cells UT937 and Thp-1, although this signal could be due to nonspecific binding. Policealne antibodies weakly percentno respond to mouse b-cell line A20, but the authors did not observe staining of murine thymoma EL4.

Four monoclonal antibodies to zalpha11 (example 35) conjugatively with Biotin and a subpopulation of cells described above were subjected to screening for the expression of zalpha11 receptor (BaF3, BaF3/zalpha11, Raji, Jurkat and resting HL-60). Cells were collected, about Yali, then resuspendable in 100ál WB, containing 15 μg/ml of one of the 4 biotinylated mAb. Cells were incubated with mAb for 20 minutes on ice, then washed with 1.5 ml WB and precipitated in a centrifuge. The supernatant was removed by aspiration and precipitation resuspendable 100 ál CyCrome-conjugated streptavidin (CYC-SA; PharMingen)and then incubated on ice for 20 minutes and washed and precipitated as described previously. Control tubes contained cells that stained only WM-SA. Precipitation resuspendable in 400 μl of WB and flow cytometry were performed as described above. Positive staining was determined in the form of a signal exceeding the level of background staining one CyC-SA. Application F3/zlh11-transfectant as control allowed the authors to build these 4 mAb in connection with their corresponding mean fluorescence intensities (MFI), which may reflect the affinity of the antibody and/or the degree of biotinidase mAb. These mAb were as follows, from highest to lowest MFI: 249.28.2.1.2.2; 247.10.2.15.4.6; 249.19.2.2.3.5 and 249.15.2.4.2.7. The Raji cells stained positively mAb zalpha11. The Jurkat cells were positively stained mAb zalpha11, but not as much as on b cells (Raji). Thus, zalpha11 receptor expressively on these b - and T-cell lines. The distribution of staining in non-activated cells HL60 were identical for all of these mAb, and the signal was PTS is ery weak. The authors believe that this signal does not reflect the true expression of zalpha11 cells HL60, but rather may be caused by nonspecific binding of murine mAb with human cells, possibly through Fc-receptors.

Example 49

Action zalpha11 ligand on b cells and protein zalpha11 ligand-toxic saporin

Steps zalpha11 ligand person was tested on the following In-human cell lines: cell lines lymphoma, Burkitt's lymphoma Raji (ATCC No. CCL-86) and Ramos (ATCC No. CRL-1596); cell line b-cell lymphoma EBV person RPMI 1788 (ATCC No. CRL-156); line IM-9 myeloma plasmacytoma person (ATCC No. CRL159); and the EBV-transformed b-cell line DAKIKI person (ATCC No. TIB-206) and cell line HS Sultan (ATCC No. CRL-1484). After approximately 2-5 days processing zalpha11-ligand changes in the expression of the marker was detected in the cell line IM-9, Raji, Ramos and RPMI1788, suggesting that these cells respond to zalpha11 ligand. In cell lines of human, processed zalpha11-ligand, grow much more slowly than untreated cells when re-seeding in the Cup for cell culture. These cells also had increased expression of Fas-ligand, as determined by flow cytometry (example 49D and example 49E) and moderately increased sensitivity to activating Fas-antibody (example 49A). This result indicates that zalpha11 ligand could control some ti is s In-cell neoplasm induced their differentiation in less proliferative and/or more Fas-ligand-sensitive state. In addition, zalpha11 receptor is expressed on the surface of some of these cell lines (see example 48). Thus, zalphall-ligand and conjugate zalpha11 ligand human immunotoxin saporin (example 49B, below) or other protein zalpha11 ligand-toxin could be used in therapy in b-cell leukemia and lymphoma.

A. Action zalpha11 ligand man on b-cell line

Cells IM-9 were sown at approximately 50,000 cells/ml +/- 50 μg/ml purified zalpha11 ligand person (example 29). After 3 days of growth the cells were harvested, washed and counted, and then re-sown at approximately 2500 cells/ml in 96-well plates in wells with 0 0,033, of 0.1 or 0.33 mg/ml of antibodies against FAS (R&D Systems, Minneapolis). After 2 days spent fluorescent assay with Alamar blue (example 2B) to assess proliferation of these cells.

Processed zalpha11-ligand cell IM-9 grew only 27% of the density of untreated cells in the absence of antibodies anti-FAS. In the presence of 0.33 µg/ml antibody anti-FAS treated zalpha11-ligand cells inhibited by an additional 52%, whereas untreated cells inhibited only 30%. Total inhibition of cell growth treatment as zalphall-ligand, and 0.33 mg/ml of antibody anti-FAS was 86%.

When pretreatment of the cells IM-9 within three days zalpha11-ligand or without zalpha11 ligand and subsequent re-seeding PR is 100 cells per well and grown with the antibody anti-FAS or without antibodies anti-FAS within 6 days of growth in untreated cells, evaluate test with Alamar blue (example 2B), inhibited only 25% of the antibody anti-FAS, whereas the growth of processed zalpha11-ligand cells inhibited by 95% relative to the growth of untreated cells in the absence of antibodies anti-FAS.

Century, the merge Action zalpha11 ligand human immunotoxin saporin on b-cell line

Construction and purification of the conjugate zalpha11 ligand human immunotoxin saporin (zalpha11L-sap) described in example 50. Zalpha11L human sap was much more potent than one saporin in the inhibition of cell growth. When re-seeding of treated cells after three to four days of treatment with conjugate Zalpha11L man-sap cells grew very poorly.

Cells IM-9, Ramos and C ATS No. CCL-243) were sown at approximately 2500 cells per well in 96-well tablets 0-250 ng/ml conjugate Zalpha11L man-sap or only with 0-250 ng/ml saporin (Stripe et al., Biotechnology 10:405-412, 1992) as a control. The plates were incubated for 4 days, then did an analysis of proliferation with Alamar blue (example 5B). When the maximum concentration of conjugate huzalpha11L-sap growth of cells in IM-9 cells and Ramos inhibited by 79 and 65%, respectively. Cells C that are low/negative according to flow cytometry in relation to the expression of zalpha11 receptor, had no effect zalpha11L-sap, indicating specificity of action of the conjugate.

Cells IM-9 were sown 5000 cells/ml in 6-hole cups at 0 and 50 ng/ml conjugate zalpha11L human sap. After 3 days cells were collected and counted and then sown from 100 to 0.8 cells per well in 2-fold serial dilutions and 12 holes on one cell cultivation without conjugate zalpha11 ligand human immunotoxin saporin. After 6 days, the number of wells with growth at each cell dilution was assessed in accordance with the results of the analysis of proliferation with Alamar blue (example 2B).

When evaluating the number of cells by means of analysis with Alamar blue (example 2B) after 6 days of growth of the control cells, seeded at approximately 12.5 and 6.25 cells per well, had equivalent growth treated with zalpha11-sap cells, seeded at 100 and 50 cells per well, respectively. Thus, the growth of survivors treated cells IM-9 was significantly weakened even after uninstalling through reseeding conjugate zalpha11-sap immunotoxin.

Limited tissue distribution zalpha11 receptor of the person (example 48) and the specificity of action of zalpha11-sap in relation expressing the receptor cell lines suggest that this conjugate may be tolerated in vivo.

C. the conjugate Action zalpha11 ligand human immunotoxin saporin on the viability of b-cell line

Cells HS Sultan (ATCC No. CRL-1484) were sown at about 40,000 cells per ml in 12-hole plates and were grown for five days or without the addition of cytokines or 4 0 ng/ml purified zalpha11-leagues the NDA person (example 29) or 25 ng/ml conjugate huzalpha11L-sap (example 50 below) or with 20 ng/ml IFN-alpha (RDI) or zalpha11 ligand and IFN-alpha. ZalphaH-ligand inhibited the growth of cells HS Sultan 63%. IFN-alpha inhibited growth of 38%. Zalpha11 ligand plus IFN-alpha inhibited growth of 78%, indicating that the inhibiting effects zalpha11 ligand person and IFN-alpha can be additive. Zalpha11L-saporin(sap) inhibited cell growth HS Sultan 92%.

The results above confirm the possible use zalpha11 ligand or zalpha11L human sap in the treatment of zlokacestvennosti or other zabolevanii that Express zalpha11 receptor, in particular diseases of b-cell origin. Combining zalpha11 ligand with IFN-alpha is particularly expected due to their additive action in the inhibition of cell HS Sultan. Some other types of lymphoid zlokacestvennosti and diseases can also Express zalpha11 receptor as activated T cells also Express mRNA of the receptor (example 48), and some of these diseases may be more sensitive to zalpha11-ligand in therapy using merge zalpha11 ligand-toxin.

D. FAS (SR)-expression In human cell lines is increased by stimulation of the zalpha11-ligand person

In cell lines of human HS-Sultan (ATCC No. CRL-1484), IM-9 (ATSS No. CRL159), RPMI 8226 (ATSS No. CCL-155), RAMOS (ATCC No. CRL-1596), DAKIKI (ATCC No. TIB-206) and RPMI 1788 (ATCC No. CRL-156) was treated with 10-50 ng/ml of purified zalphall-ligand person (or without it) (example 29) to use the e 2-8 days. Then these cells were stained with PE-conjugated antibody anti-C (PharMingen, San Diego, CA) according to the manufacturer's Protocol and analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA). In all cell lines staining anti-S (FAS or APO-1) was increased in some cases by more than 2 times when handling zalpha11-ligand person.

That is, FAS (SR)-expression in primary murine b cells of the spleen increases the stimulation of the zalpha11-ligand person

Primary murine splenocytes were obtained by crushing spleens from 8-12-week-old mice C57/BL6. Erythrocytes were literally processing of the drug for 5 seconds with water and then passed through a sieve of 70 μm. The remaining splenocytes were washed and were sown in RPMI medium (JRH Bioscience) plus 10% HIA-FCS (Hyclone, Logan, UT). Interleukin-2 (IL-2) (R&D Systems) c zalpha11-ligand person or without zalphall-ligand person, as described above. Then they were incubated at 37°With 5% CO2within 5 days. Splenocytes were collected and stained with PE-conjugated antibody anti-D95 (PharMingen) and FITC-conjugated antibody anti-D19 (PharMingen) according to the manufacturer's Protocol. Cells were analyzed by flow cytometry on a FACScalibur (Becton Dickinson). When installing discriminatory Windows on mouse CD19+ b-cells was found that the staining antibody anti-D95 was increased In cells treated with IL-2 plus zalpha11 ligand person in comparison to the situation with staining in the same IL-2. Anti-D95-staining was equal to 37 relative fluorescent units (RFU) on b cells in a single IL-2 and 55 RFU on b cells cultured in IL-2 and zalpha11-ligand person.

Example 50

Construction and purification of fusion zalpha11 ligand-toxin

The contract on delivery 10 mg zalpha11 ligand person (example 29) was sent to Advanced Targeting Systems (ATS, San Diego, CA) for conjugation with plant toxin-caporino (Stripe et al., Biotechnology 10:405-412, 1992). Zymogenetics received from ATS 1.3 mg protein conjugate containing 1.1 molecules saporin molecule zalpha11 ligand person prepared at a concentration of 1.14 mg/ml in 20 nm sodium phosphate, 300 nm sodium chloride, pH 7,2.

Example 51

Merge zalpha11 ligand-toxin in vivo

A. the Test conjugate zalpha11-saporin in mice

Conjugate zalpha11-saporin (example 49) was administered to C57BL6 mice (females, age 12 weeks, purchased from Taconic) in two different doses: 0.5 and 0.05 mg/kg, Injection, iv spent in medium consisting of 0.1% BSA (ICN, Costa Mesa, CA). Three injections were performed during one week (day 0, 2 and 7). Blood samples were taken from mice at day 0 (before injection) and on days 2 and 8 (after injection). Blood was collected in heparinized tubes (Becton Dickinson, Franklin Lakes, NJ) and the number of cells was determined using an automatic hematological analyzer (Abbot Cell-Dyn model No. CD 3500CS, Abbot Park, IL). Animals were autonational and autopsical in day 8 sat after the RA blood. Spleen, thymus, liver, kidney and bone marrow were removed for histopathology. Spleen and thymus were weighed and an additional blood sample was collected in tubes to separate the serum. Serum was sent to Pheonix Central Labs, Everett, WA for testing in the standard chemical panel. Samples were also collected for flow cytometrical analysis, as described here.

Formula circulating blood count (hemogram) and measurement of serum chemistry did not differ significantly between treated conjugate zalpha11 ligand mice and mice treated with an equivalent dose of unconjugated toxin (saporin). Histological analysis of the tissue in the treated conjugate zalpha11 ligand-saporin mice did not show significant changes relative to mice treated with an equivalent dose of unconjugated toxin. These results show that the conjugate saporin was not toxic in vivo.

C. Test conjugate zalpha11-toxin saporin at the events of b-cell tumors in vivo

Steps zalpha11 ligand man and merge zalpha11 ligand man-toxin saporin (example 50) on tumor cells was tested in vivo using a murine model of tumor xenotransplant described here. Xenograft models were first tested using cell lines, selected on the basis of in vitro experiments, such as lines, described what's in example 49. These cell lines include, but are not limited to, cell lines lymphoma Burkitt human Raji (ATCC No. CCL-86) and Ramos (ATCC No. CRL-1596); cell line human RPMI 1788 (ATCC No. CRL-156); line cell myeloma/plasmacytoma human IM-9 (ATSS No. CRL159); cell line human DAKIKI (ATCC No. TIB 206) cells and HS Sultan (ATCC No. CRL-1484). Cells derived directly from human cancers, can also be used in this type of model. In this way, the screening of patient samples for sensitivity to processing zalpha11-ligand or conjugate zalpha11 ligand-toxin saporin can be used to select the optimal indications for the use of zalpha11 in anticancer therapy.

After selection of a suitable model in vivo xenograft, as described above, zalpha11 ligand-induced activity of natural killer cells (NK-cells) and/or actions zalpha11 ligand on the tumor cell of origin appreciate in vivo. Zalpha11 ligand person have on its ability to generate cytotoxic effector cells (for example, NK-cells) with activity against tumors of b-cell origin with the use of murine models of tumor xenografts, as described here. In addition, there may be direct actions zalpha11 ligand of the tumor. Models of transplant for the experiments was chosen, as described above. FR is the number of applications stimulated zalpha11-ligand in human cells develop and test the effectiveness of the depletion of tumor cells and stimulation of survival in mice inoculated cell lines or primary tumors.

Example 52

Identification of clones artificial chromosomes P1 containing genomic DNA zalpha11 ligand person

cDNA-insert zalpha11 ligand man amplified using PCR using primers based on the vectors. The PCR product32R-were labeled and hybridized to the filters of high density, representing RACES library (Artificial chromosomes P1). Filters and frozen source solutions library was obtained from Roswell Park Cancer Institute, Buffalo, New York; the segment library was a RPC16 with 4-fold depth of coverage. Filters are hybridized overnight at 65°C in ExpressHyb (Clontech) and washed according to the manufacturer's recommendations. Transcript of positive signals has led to the identification of four RACES-clones, named N, A, 105G9 and N. PCR analysis using primers specific for the 5'-end (ZC22452 (SEQ ID NO:100) and ZC22451 (SEQ ID NO:101)) and 3'-end (ZC22450 (SEQ ID NO:102) and ZC22449 (SEQ ID NO:103)) the coding region showed that RAS A and RACES 105G9 contained both ends, whereas RACES N and RACES N contained only the 5'-end. RACES N were digested with Eco RI and Not I and identified fragment 9 TPN that hybridized with cDNA probe zalpha11 ligand. This fragment was isolated and was subclinically using the methods described here in pBluescript II SK(+) (Stratagene), tentative is but cleaved with Eco RI and Not I. Sequencing revealed that this fragment contained approximately 380 BP promoter region, exons 1, 2 and 3, full introns 1 and 2 and ending in intron 3.

the 3'end of the gene zalpha11 ligand person received via PCR using DNA from RACES A as template, with primers ZC23771 (SEQ ID NO:104) and ZC22449 (SEQ ID NO:103). Used DNA Taq polymerase with supplied buffer with the addition of 4% DMSO. Reaction conditions were as follows: 94°C, 5 minutes; followed by 35 cycles of 94°C for 30 seconds, 52°C for 1 minute, 72°C for 3 min; then 72°C for 7 minutes. This gave a fragment of 2.9 TPN, which contains part of exon 3, full introns 3 and 4, the complete exon 4 and the coding part of exon 5.

Genomic structure of the gene zalpha11 ligand person is following from 5' to 3': SEQ ID NO:105, containing about 240 BP of the promoter, exon 1 (nucleotides 240-455 SEQ ID NO:105), intron 1 (number of nucleotides 456-562 SEQ ID NO:105), sksan 2 (number of nucleotides 563-598 SEQ ID NO:105) and part of intron 2 containing 5' 748 BP (nucleotides 599-1347 SEQ ID NO:105); a gap of approximately 3 TPN; SEQ ID NO:106, containing 3' 718 BP of intron 2, exon 3 (nucleotide 719-874 SEQ ID NO:106) and part of the 5'-end of intron 3 (number of nucleotides 875-1656 SEQ ID NO:106); the gap is less than approximately 800 BP; SEQ ID NO: 107, containing 644 BP of intron 3; gap less than approximately 800 BP; SEQ ID NO:108, containing the 3' 435 P.N. Intro is 3, exon 4 (nucleotide 436-513 SEQ ID NO:108), intron 4 (number of nucleotides 514-603 SEQ ID NO:108) and part of the 5'-end of exon 5 (number of nucleotides 604-645 SEQ ID NO:108).

Example 53

Research associate125I-labeled zalpha11 ligand human cell lines

25 micrograms of purified zalphall-ligand person (example 29) were labeled with 2 MCI125I use iodophenol (Pierce, Rockford Illinois) according to the manufacturer's instructions. This labeled protein was used to assess binding zalpha11 ligand person with Raji cells human (ATSS No. CCL-86) using binding to murine cells wild-type BaF3 and BaF3 cells, transfitsirovannykh zalpha11 receptor (BaF3 cells/hzalpha11), as controls. Expected binding zalpha11 ligand with cells BaF3/hzalpha11 (positive control), while not expected to bind to the BaF3 cells wild-type (negative control), based on the results of the analysis of proliferation (example 5). About 5×105of Raji cells per well, 1×106BaF3/hzalpha11 and 1×106the BaF3 cells per well were sown separately in 96-well plates. Ten ng/ml of labeled zalpha11 ligand person was added in duplicate to the wells with serial dilutions of unlabeled competitor-zalpha11 ligand person added from a 250-fold molar excess in dilutions of 1:4 to 0,061-fold molar excess. Each point is analyzed in two replicates. After adding labeled zalpha11 ligand man to the hole they had made incubated at 4°C for 2 hours, to allow the ligand to contact with these cells. Then cells were washed 3x in the buffer for binding (RPMI-1710 (JRH Bioscience) with 1% BSA (Sigma)and counted on a gamma counter COBRA II AUTO-GAMMA (Packard Instrument Company, Meriden, CT).

The binding of labeled zalpha11 ligand with cells was observed in Raji cells and BaF3/hzalpha11. In addition, in the case of Raji cells on average 250-fold molar excess of unlabeled zalpha11 ligand reduced the binding of 3 times in the presence of unlabeled nonspecific competitor (Interferon Gamma from R&D Systems, Minneapolis, MN) and 3.7 times compared to the variant without a competitor. Observed dose-dependent competition for specific unlabeled competitor, zalpha11 ligand person. Thus, the binding of zalpha11 ligand with Raji cells was specific. Similarly, for a positive control, cells BaF3/zalpha11, 250-fold molar excess of unlabeled zalpha11 ligand reduced the binding of 2 times relatively nonspecific competitor and a 3.06 times on option without a competitor. Thus, the binding of zalpha11 ligand with cells BaF3/zalpha11 was also specific. Not watched competitive binding with BaF3 cells of the wild type. Thus, it was shown that zalpha11 ligand specifically binds to Raji cells and the cell is mi BaF3/hzalpha11, but not with cells of the negative control BaF3.

Example 54

Expression of zalpha11 receptor on human blood cells

A. isolation and culturing of cells in human peripheral blood

Viewsat human blood was diluted 1:1 using SFR (Gibco BRL) and were layered on Ficoll/Hypaque Plus (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) and was turned off for 30 minutes at 1800 rpm and allowed to stop the centrifuge without braking. The interfacial layer was removed and transferred into a fresh tube Falkon 50 ml (Falcon, VWR, Seattle, WA), brought to a final volume of 40 ml SPR and off for 10 minutes at 1200 rpm with the included brake. The viability of the selected cells was tested with Trypanosoma blue (Gibco BRL) and cells resuspendable at a final concentration of 1×106cells/ml cell medium (RPMI medium 1640, 10% inaktivirovannaja heating fetal calf serum, 5% L-glutamine, 5% pen/strap) (Gibco BRL).

Cells were cultured in 6-hole plates (Falcon, VWR) for 0.4 or 24 hours with a variety of the following incentives. Stimulation of anti-lgM, anti-S and anti-CDS was performed as in example 44 and example 42. Formalparameterlist (PMA) and ionomycin (Sigma, St. Louis, MO) (example 5C) was added to appropriate wells at 10 ng/ml and 0.5 mg/ml, respectively. Cells were incubated at 37°With humid thermostat for varying periods of time.

C. Staining with antibodies and analysis

Cells were collected from cups, washed and resuspendable in chilled on ice coloring medium (HBSS, 1% fetal calf serum, 0.1% of sodium azide) at a concentration of about ten million cells per milliliter. Blocking of Fc-receptor and non-specific binding of antibodies to the cells was achieved by adding 10% normal goat serum (Gemini Bioproducts, Woodland, CA) and 10% normal human serum (Ultraserum, Gemini) to the cell suspension. Aliquots of cell suspensions were mixed with FITC-labeled monoclonal antibody against a marker of differentiation of CD3, CD19 or CD14 (PharMingen, La Jolla, CA) and biotinylated monoclonal antibody against zalpha11 receptor human (huzalpha11) (example 35). Specificity of staining was determined by competition using rasterimage receptor zalpha11 CEE (example 10A) with a 10-fold excess by weight. After incubation on ice for 60 minutes the cells were washed twice in chilled on ice coloring medium and resuspendable in 50 ml of staining medium containing streptavidin-PE (Caltag, Burlingame, CA). After 30-minute incubation on ice, cells were washed twice in chilled on ice proryvnym buffer (STR, 1% calf serum, 0.1% of sodium azide) and resuspendable in the washing buffer containing 1 mg/ml 7-AAD (Molecular Probes, Eugene, OR) as a marker innespace the property. Data flow cytometry was received on living cells using a flow cytometer FACSCalibur (BD Immunocytometry Systems, San Jose, CA). Both queries and data analysis were performed using CellQuest software (BD Immunocytometry Systems).

The results of staining with antibody anti-zalpha11 showed that zalpha11 receptor of the person is expressed on peripheral blood cells of a person expressing CDS, CD19 or CD14. Staining for CD3 and CD19 cells was specific, as evidenced by the absolute competition with soluble zalpha11 receptor. Staining for CD14 cells found some specificity in respect of this ligand, as evidenced by partial competition with soluble receptor. Activation or T-cell antibody anti-CD3, or b-cell antibody anti-CD40 resulted in an increased level of zalpha11 cell surface in 24 hours. Did not observe any increase in the level of expression of zalpha11 at 4 hours with any incentive to any cell population. Treatment of cells zalpha11-ligand resulted in reduced zalpha11-staining on CD3-positive and CD19-positive cells, but not in CD14-positive cells at 4 and 24 hours.

Example 55

Preliminary assessment of water stability zalpha11 ligand person

Conducted preliminary studies to assess the characteristics of water stability zalpha11 ligand, chelovekov maintaining Bioprocessing, preparation of compositions and injection in vivo. The following objectives: 1) to check the stability and extraction of minnesoto Alzet Minipumps and under normal storage and manipulation, 2) the definition of detecting the stability of some analytical methods, including cation exchange VIH (CX-HPLC), obremenitve VIH (RP-HPLC), gel-filtration VIH (SEC-HPLC) and bioanalysis (F3/zlh11R-proliferation (e.g., example 2 and example 4), and 3) determination of limiting the stability of the degradation pathways and their kinetic dependencies.

Aliquots of purified zalpha11 ligand person (example 29) were prepared by dilution to 2 mg/ml in SFR (pH 7.4) and kept in cryoplane from polethylene low density (LDPE) (Nalgene, 1.8 ml) at -80°With (control), 5°, 30°37°C. the Samples were analyzed periodically for 29 days using the CX-, RP-, SEC-HPLC and bioanalysis. Aliquots were also kept at -80°and were subjected to cyclic freezing-thawing (n/a) (-80°C/RT; 5x C/o 10x n/a). Recovery to normal zalpha11 ligand man was determined in comparison with control -80°C (1 C/o) in all analyses.

The remaining solution zalpha11 ligand person from the control -80°-re-samples were frozen (-80° (C) after analysis. This aliquot (2 C/o) was used to assess thermal and conformational stability zalpha11 ligand person as f is NCLI pH using circular dichroism (CD). A solution of 2 mg/ml was diluted to 100 μg/ml in P-buffers in the pH range of 3.3 to 8.8. CD spectra in the far UV subjected to monitoring for temperature range 5-90°With intervals 5° (n=3/pH). Used CD spectropolarimeter has been created Jasco 715 (Jasco, Easton, MD). Thermal deployment molecules were subjected to monitoring changes in the ellipticity at 222 nm as a function of temperature. Evaluation of Tmreceived under the assumption deployment model with two States. Data built using software SlideWrite Plus for Windows v4.1 (Advanced Graphics Software; Encinitas, CA).

The extraction and stability of minnesoto Alzet Minipumps (Model No. 1007D; ALZA Corporation, Mountain View, CA) was evaluated by filling pumps 100 μl solution of 2 mg/ml zalpha11 ligand person, location of pumps 1.8 ml LDPE containing 1 ml SFR (pH 7.4) and stored them at 37°C. Releasing/removing the zalpha11 ligand person from minnasota was assessed using the CX-, RP-, SEC-HPLC on days 2, 4 and 7. Activity was assessed using bioanalysis in day 7. This study is intended to assess the release of 3 pumps during sampling.

Chromatographic data suggest that SH - and SEC-HPLC (cation exchange VGH and gel filtration VIH) were detecting stability, while obremenitve VIH (RP-HPLC) did not possess this property. At least 3 additional peak pointing to visible degradation products, observed when using CX-HPLC. SEC-HPLC (gel-filtration VIH) divided the visible units zalpha11 ligand person, eluruumina before zalpha11 ligand person. However, not observed significant additional peaks, eluruumiks after the peak zalpha11 ligand person. This suggests that the degradation products observed using cation exchange VIH (CX-HPLC)occur, most likely because of modifications of amino acids, such as deliciousa, and not from the processes of hydrolysis/proteolysis, leading to truncated variants. A small amount of blur front/education "tails" observed when obremenitve VIH (RP-HPLC) (relative to control) in the samples, which, as shown, has undergone significant degradation according to gel-filtration (SEC-HPLC) and cation exchange VIH (CX-HPLC). However, the visible degradation products were not divided obremenitve VIH (RP-HPLC). The degradation observed in cation exchange VIH (CX-HPLC), increased as a function of time-temperature and was accompanied by an apparent first order kinetics. % zalpha11 ligand person extracted using cation exchange VIH (CX-HPLC) after 29 days at 37°, 30°and 5°was 39%, 63% and 98%, respectively. Aggregation was also increased dependent on the time-temperature. percent of the unit found in the preparations stored for 29 days at 37°, 30°and 5°With, who was b 7,4, 3,4 and below detectable limits (BDL), respectively. Did not observe significant differences using bioanalysis in any sample, suggesting that the degradation products had activity equivalent to the activity of intact zalpha11 ligand person. No observed degradation in any analysis in samples subjected to 10 cycles C/O.

The release of zalpha11 ligand man from Alzet Minipumps is consistent with theoretically expected volume release. This suggests that significant surface adsorption does not impair the delivery of zalpha11 ligand person using Alzet Minipumps with concentration when filling 2 mg/ml was Observed degradation, consistent with previously celebrated degradation. Purity in %, determined using cation exchange VIH zalpha11 ligand man released after 2, 4 and 7 days was 96, 90 and 79%, respectively. It should be understood that the degradation takes place after zalpha11 ligand person is released into the environment the release or diluted with a medium release. Thus, the % purity in Minnesota may be slightly different from the %specified in the environment of the release. The bioactivity of each sample is consistent with the expected number of zalpha11 ligand person released from minnasota.

CD spectra in the short wavelength (far) UV-region zalph11-ligand person, as expected, they are compatible with interleukins, such as IL-3 (J. Biochem. 23:352-360, 1991), IL-4 (Biochemistry, 30:1259-1264, 1991) and IL-6 mutant (Biochemistry, 35:11503-11511, 1996). Did not observe large changes in the far UV region of the CD spectra as a function of pH. The results showed that the pH of maximum thermal stability/conformational stability was approximately pH 7.4. Analyses curves deployment molecules were based on the mechanism of two States of deployment in order to make possible the comparison of thermostability/conformational stability depending on pH/composition. However, there may be one or more intermediate products during the deployment process, as cooperatively was relatively low based on nehluboko curve deployment. Although special studies to determine whether re-laying zalpha11 ligand person after thermal deployment of up to 90°has not been conducted, preliminary data suggest that at least partial re-laying occurs after the return temperature of the sample to 20°With cooling.

These studies allow us to identify analytical example for cleaning and checking the stability of the zalpha11 ligand person. For example, gel filtration WICH can be used to characterize the extent and rate of aggregation in aqueous solution is. Similarly, cation exchange WICH can be used to characterize the extent and rate of degradation of zalpha11 ligand person by mechanisms other than aggregation. The bioanalysis you can use to test the activity of zalpha11 ligand man and his products water degradation. For example, variants of zalpha11 ligand person obtained in aqueous solution and separated using cation exchange VIH (CX-HPLC), can themselves be used as therapeutic agents, since they have equivalent biological activity. The fact that zalpha11 ligand person are degraded in a number of different processes (aggregation, modifications of amino acids), suggests that the preferred or unique composition, which minimizes the rate of degradation, it may be necessary for long-term stability of the product in solution.

Identification of the nature of the product water degradation and determination of their kinetic dependencies (pH, concentration, fillers) are in the process of research. Stability zalpha11 ligand human serum/plasma is determined to confirm the planning and interpretation of in vivo studies.

From the foregoing it should be clear that, although the characteristic variations of the present invention is described here for the purpose of illustration, various modifications may be made by without deviating from the idea and scope of this invention. Thus, this invention is not limited by anything except the supplied formula .z

1. Selected zalpha11 polypeptide-ligand containing a sequence of amino acid residues that is at least 90% identical to residues 41 (Gln) to 148 (Ile) in SEQ ID NO:2, and the residue at position 44 is Asp, the residue at position 47 is Asp and the residue at position 135 is Glu, and the polypeptide binds zalpha 11 receptor shown in SEQ ID NO:115.

2. The selected polypeptide according to claim 1, where the amino acid residues 71, 78, 122 and 125 are cysteine.

3. The selected polypeptide according to claim 1, where the sequence of amino acid residues of at least 95% identical to SEQ ID NO:2 from residue 41 (Gln) to residue 148 (Ile).

4. The selected polypeptide according to claim 1, where the sequence of amino acid residues is 100% identical to SEQ ID NO:2 from which the STATCOM 41 (Cln)to residue 148 (Ile).

5. Selected zalpha11 polypeptide-ligand containing a sequence of amino acid residues shown in SEQ ID NO:2 from residue 32 (Gln) to residue 162 (Ser) or from residue 1 (Met) to residue 162 (Ser).

6. The selected epitope zalpha11 ligand that contains at least 14 contiguous amino acid residues of SEQ ID NO:2.

7. The selected epitope according to claim 6, where these amino acid residues selected from the group consisting of:

(a) amino acid residues 41-56 SEQ ID NO:2;

(b) amino acid residues 69-84 SEQ ID NO:2;

(c) amino acid residues 92-105 SEQ ID NO:2, and

(d) amino acid residues 135-148 SEQ ID NO:2.

8. Protein zalpha11 ligand containing at least four polypeptide, where the order of polypeptides from N-Terminus to the C-end is as follows:

the first polypeptide that includes the sequence of amino acid residues from 41 to 56 of SEQ ID NO:2;

the first spacer of 6-27 amino acid residues;

a second polypeptide that includes the sequence of amino acid residues selected from the group consisting of:

(a) residues 53-75 SEQ ID NO:111 spiral In IL-2;

(b) residues 65-83 SEQ ID NO:112 spiral In IL-4;

(c) residues 84-101 SEQ ID NO:113 spiral In IL-15;

(d) residues 72-81 SEQ ID NO:114 spiral In GM-CSF and

(e) amino acid residues 69-84 SEQ ID NO:2;

a second spacer of 5-11 amino acid residues

third polypeptide that contains a sequence of amino acid residues selected from the group consisting of:

(a) residues 87-99 SEQ ID NO:111 spiral With IL-2;

(b) residues 95-118 SEQ ID NO:112 spiral With IL-4;

(c) residues 107-119 SEQ ID NO:113 spiral With IL-15;

(d) residues 91-102 SEQ ID NO:114 spiral With GM-CSF and

(e) amino acid residues 92-105 SEQ ID NO:2;

a third spacer of 3-29 amino acid residues and a fourth polypeptide that contains a sequence of amino acid residues selected from the group consisting of:

(a) residues 103-121 SEQ ID NO:111 helix D of IL-2;

(b) residues 134-157 SEQ ID NO:112 helix D of IL-15;

(c) residues 134-160 SEQ ID NO:113 helix D of IL-4;

(d) residues 120-131 SEQ ID NO:114 helix D of GM-CSF and

(e) amino acid residues 135-148 SEQ ID NO:2.

9. The selected polynucleotide molecule encoding a zalpha11 ligand containing a nucleotide sequence corresponding to the amino acid sequence of the polypeptide according to claim 1.

10. The selected polynucleotide molecule according to claim 9, where these nucleotides are as shown in SEQ ID NO:1 from nucleotides 167 to nucleotide 490, or such as shown in SEQ ID NO:3 from nucleotide 121 to nucleotide 444.

11. Selected polynucleotide that encodes a polypeptide, which is the epitope zalpha11 ligand and containing a nucleotide sequence, the corresponding amino acid sequence of the polypeptide of claim 6.

12. Selected polynucleotide that encodes a polypeptide, which is the epitope zalpha11 ligand and containing a nucleotide sequence corresponding to the amino acid sequence of the polypeptide shown in SEQ ID NO:2 from residue 32 to residue 162.

13. Selected polynucleotide indicated in paragraph 12, where these nucleotides are nucleotides shown in SEQ ID NO:1 from nucleotide 140 to nucleotide 532, or nucleotides shown in SEQ ID NO:3 from nucleotide 94 to nucleotide 486.

14. Selected polynucleotide that encodes a polypeptide, which is the epitope zalpha11 ligand and containing a nucleotide sequence corresponding to the amino acid sequence of the polypeptide shown in SEQ ID NO:2 from residue 1 to residue 162.

15. Selected polynucleotide at 14, where these nucleotides are nucleotides shown in SEQ ID NO:1 from nucleotide 47 to nucleotide 532, or nucleotides shown in SEQ ID NO:3 from nucleotide 1 to nucleotide 486.

16. A method of obtaining antibodies to the polypeptide zalpha11 ligand involving the inoculation of an animal with the polypeptide selected from the group consisting of:

(a) a polypeptide consisting of 9-131 amino acids, where this polypeptide is identical to a contiguous sequence of amino acid residues in SEQ ID NO:2 from the number of amino acids 32 (Gln) to amino acids 162 (Ser);

(b) polypep the IDA according to claim 1;

(c) a polypeptide containing the amino acid sequence of SEQ ID NO:2 from the number of amino acids 41 (Gln) to amino acids 148 (Ile);

(d) a polypeptide containing the amino acid sequence of SEQ ID NO:2 from the number of amino acids 41 (Gln) to amino acid 56 (Val);

(e) a polypeptide containing the amino acid sequence of SEQ ID NO:2 number 69 amino acids (Thr) to amino acid 84 (Leu);

(f) a polypeptide containing the amino acid sequence of SEQ ID NO:2 from number 92 amino acids (Asn) to amino acid 105 (Arg);

(g) the polypeptide containing the amino acid sequence of SEQ ID NO:2 number 135 amino acids (Glu) to amino acids 148 (Ile);

(h) the polypeptide containing the amino acid sequence of SEQ ID NO:72;

(i) a polypeptide containing the amino acid sequence of SEQ ID NO:73;

(j) a polypeptide containing the amino acid sequence of SEQ ID NO:2 from the number of amino acids 32 (Gln) to amino acids 162 (Ser);

(k) the polypeptide containing the amino acid sequence of SEQ ID NO:2 from the amino acid 1 (Met) to amino acids 162 (Ser);

(1) a polypeptide containing the amino acid sequence of SEQ ID NO:2 from the number of amino acids 114 to amino acid 119;

(m) a polypeptide containing the amino acid is an explicit sequence of SEQ ID NO:2 from the number of amino acids 101 to the number of amino acids 105;

(n) a polypeptide containing the amino acid sequence of SEQ ID NO:2 from the number of amino acids 126 to amino acids 131;

(o) a polypeptide containing the amino acid sequence of SEQ ID NO:2 from the number of amino acids 113 to amino acid 118; and

(b) a polypeptide containing the amino acid sequence of SEQ ID NO:2 number 158 amino acids to amino acids 162;

moreover, the specified polypeptide induces an immune response in the animal with the formation of antibodies; and

the selection of this antibody from the animal.

17. Antibody zalpha10-ligand that is specific linked with zalpha11-ligand and obtained by the method according to item 16.

18. Selected zalpha11 polypeptide-ligand containing a sequence of amino acid residues shown in SEQ ID NO:56 from residue 23 (Gln) to residue 146 (Ser) or from residue 1 (Met) to residue 146 (Ser).



 

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