MXPA98005946A - Chemistry genes cx3c from mamif - Google Patents

Abstract

The present invention relates to nucleic acids encoding a new family of chemokines, the CX3C family, from a mammal, the reagents related thereto, including specific antibodies and purified proteins. Methods for using reagents and related diagnostic kits are also provided

Description

CHEMISTRY GENES CX3C MAMIFER The present invention is a continuation in part of the co-pending US Application Serial No. 08 / 849,006, filed May 16, 1996, which is a continuation in part of the US Application Serial No. 08 / 590,828. Submitted on January 24, 1996, which are incorporated herein by reference.

FIELD OF THE INVENTION The present invention contemplates compositions related to proteins that function to control the development, differentiation, trafficking and physiology of mammalian cells, for example, the cells of a mammalian immune system. In particular, proteins that regulate or give evidence of the development, differentiation and function of various cell types, including hematopoietic cells, are provided.

BACKGROUND OF THE INVENTION The circulating component of the circulatory system of mammals consists of various cell types, including the red and white cells of the rangre of erythroid and myeloid cell lineages. See, for example, Rapaport (1987) Introduction to Hematology (2d ed.) Lippincott, Philadelpia, PA; Jandl (1987) Blood: Textbook of Hematology, Little, Brown and Co., Boston, MA.; and Paul (ed.) (1993) Fundamental Immunology (3rd ed.) Raven Press, N.Y. For some time it has been known that the immune response of mammals is based on a series of complex cellular interactions, known as the "immune network". Recent research has provided new insights into the internal function of this network. Although it remains clear that much of the response, in fact, revolves around the network-like interactions of lymphocytes, macrophages, granulocytes and other cells, inraunologists now generally hold the view that soluble proteins, known as lymphokines, cytokines or monocins, play an important role in the control of these cellular interactions. In this way, there is considerable interest in the isolation, characterization and mechanisms of action of the modulating factors of the cells, an understanding of which would make significant advances in the diagnosis and therapy of numerous medical anomalies, for example, the immune system and others. disorders . Lymphokines apparently mediate cellular activities in various ways. It has been shown to support the proliferation, growth and differentiation of pluripotent hematopoietic germ cells in a large number of progenitors that comprise various cell lineages that make up a complex immune system. These interactions between cellular components are necessary for a healthy immune response. These different cell lineages often respond in different ways when lymphokines are administered together with other agents. Chemokines are a large and diverse superfamily of proteins. The superfamily is subdivided into three branches, based on whether the first two cysteines in the classical motif of the chemokine are adjacent (known as the "CC" branch) or separated by an intermediate residue ("CXC"), or a new one branch lacking two cysteines in the corresponding motif, represented by the chemokines known as lymphotactins, see, for example, Schall and Bacon (1994) Current Opinion in Immulogy 6: 865-873, and Bacon and Schall (1996) Int. Arch Allergy &Immunol 109: 97-109 A large number of factors have been identified which influence the differentiation process of precursor cells or control the physiology or migration properties of specific cell types. They indicate that there are other factors whose functions in the immune function so far are unknown.These factors are responsible for the biological activities whose spectrum of effects can be different from the factors of differentiation. known activation or activation. The lack of knowledge about the structural, biological and physiological properties of the regulatory factors that regulate the physiology of cells in vivo prevents the modification of the effects of these factors. In this way, medical conditions where the regulation of the development or physiology of relevant cells is inadequate remains unmanageable.

SUMMARY OF THE INVENTION The present invention manifests the existence of a class of molecules that contain chemokine motifs previously unknown, which are hereby termed the CX3C chemokines. The CX3Ccinas have 3 amino acids that separate the cysteines in the corresponding region of the chemokine motif. Based on the analysis of the sequence of the two CX3C protein sequences described below, it is evident that the CX3Ccinas do not belong to the C, C-C, or C-X-C chemokine families. These represent the first known members of a previously unnamed new class of chemokines called CX3Ccinas or otherwise, the CX3C family of chemokines.The present invention offers a composition of matter selected from an antibody binding site that specifically binds to a mammalian CX3C chemokine, an expression vector encoding a mammalian CX3C chemokine or fragment thereof, a substantially pure protein that is specifically recognized by the antibody binding site, and a substantially pure CX3C chemokine or peptide of this, or a fusion protein consisting of a fragment of 30 amino acids of the CX3C quio cyano sequence In the embodiments of the antibody binding site, the antibody binding site can be: specifically immuno-active with a selected mature protein of the group consisting of the polypeptides of SEQ ID NO: 2, 4, 6, and 8; a purified or recombinantly produced human or mouse CX3C chemokine; in a monoclonal antibody, Fab or F (ab) 2; immunoreactive with denatured antigen; or in a labeled antibody. In certain embodiments, the antibody binding site is detected in a biological sample by a method of: contacting a binding agent having an affinity for the CX3C chemokine protein with the biological sample; incubating the binding agent with the biological sample to form a binding agent: chemokine protein complex CX3C; and detect the complex. In a preferred embodiment, the biological sample is human and the binding agent is an antibody.

A method of a kit having a composition is provided, as already described, with instructional material for the use of the composition; or the segregation of the composition in a container. One embodiment of nucleic acid of the invention includes an expression vector encoding a CX-3C chemokine protein, wherein the protein specifically binds to an antibody raised against an immunogen selected from the mature polypeptide portions of SEQ ID NO: 2, 4. , 6, and 8. The vector can: encode a CX3C chemokine polypeptide with complete sequence identity for a human CX3C chemokine domain that occurs naturally; encoding a CX3C chemokine protein comprising the selected sequence of the polypeptides of SEQ ID NO: 2, 4, 6 and 8; or comprises the selected sequence of the nucleic acids of SEQ ID NO 1, 3, 5 or 7. In other embodiments the vector is capable of selectively hybridizing to a nucleic acid encoding a CX3C chemokine protein, for example, a mature protein coding segment of SEQ ID NO. 1, 3, 5, or 7. In various preferred embodiments, the isolated nucleic acid is detected in a biological sample by a method: contacting a biological sample with a nucleic acid probe capable of selectively hybridizing to the nucleic acid; incubating the nucleic acid probe with the biological sample to form a hybrid of the nucleic acid probe with complementary sequences of the nucleic acid present in the biological sample; and determining the degree of hybridization of the nucleic acid probe in the complementary sequences of the nucleic acid. In this method, the preferred nucleic acid probe is capable of hybridizing to a nucleic acid encoding a protein containing the polypeptides of SEQ ID NO: 2, 4, 6, or 8. In the protein modalities, the chemokine protein CX3C isolated preferably will be from about 11,000 to 15,000 daltons when found in the non-glycosylated form, and the chemokine protein CX3C binds specifically to an antibody raised against an immunogen; the polypeptides of SEQ ID NO 2, 4, 6 or 8; and the CX3C chemokine lack the cysteine structural motifs and the characteristic sequence of a chemokine .C, CC or CXC. In various embodiments, the isolated CX3C chemokine protein is: selected from CX3 human CX3 or CX3 from mouse; consists of a polypeptide containing the sequence of SEQ ID NO: 2, 4, 6, or 8; it is a protein that is produced in a recombinant form or that occurs naturally. The present invention also comprises a cell transfected with the nucleic acid encoding a CX3C chemokine, for example, wherein the nucleic acid has the SEQ ID NO: 1, 3, 5 or 7. The invention also provides a method of modulating physiology or development of a cell by contacting the cell with a CX3C chemokine or a chemokine antagonist. In preferred embodiments, physiology is attracted and the cell is a peripheral blood monocyte or a T cell.

DETAILED DESCRIPTION OF THE INVENTION I. General The present invention provides DNA sequences encoding mammalian proteins which exhibit structural properties or motifs characteristic of a cytokine or chemokine. For a review of the chemokine family, see, Lodi, et al. (1994) Science 263: 1762-1767; Gronenborn and Clore (1991) Protein Engineering 4: 263-269; Miller and Kranger (1992) Proc. Nat '1 Acad. Sci. USA 89: 2950-2954; Matsushima and Oppenheim (1989) Cytokine 1: 2-13; Soteckle and Baker (1990) New Biol. 2: 313-323; Oppenheim, et al. (1991) Ann. Rev. Immunol. 9: 617-648; Schall (1991) Cytokine 3: 165-183; and The Cytokine Handbook Academic Press, NY. The proteins described herein are called CX3Cacinas because initially ._ were recognized sharing significant structural characteristics of the chemokines, but whose structural characteristics also presents peculiarity of the sequence, for example, structural motifs, different from the other known branches of chemokine molecules. The best characterized mode of this family of proteins was discovered from a human and is called human CX3C (GenBank Accession No. H14940). See, SE ID NO: 1-4. An additional CX3 cell, represented by a mouse molecule, called CX3 mouse Cina is also described herein. See ID SEC NO: 5-3. The following descriptions are directed, for example purposes, to modalities in primates and rodents, for example, human and mouse, but in the same way they are applicable to related models from other sources, for example, natural. These sources should include various vertebrates, usually warm-blooded animals, for example, birds and mammals, particularly domestic animals and primates. In the human sequence (SEQ ID NO 1-4), the signal sequence ranges from about Met to Gl? 24, in this way the mature polypeptide starts at about Gln25 and ends at about Val397. A chemokine domain ranges from about Gln25 to about Gly1OO; a stem region, which possesses multiple potential glycosylation sites, ranges from about GlylOl to about Gln341, a transmembrane region begins at about Ala342 and ends at about Thr361; and an intracellular domain, which contains two tyrosine phosphorylation sites at residues 382 and 392 ranges from about Tyr362 to Val3-9-7. In the mouse CX3C chemokine (SEQ ID NOs 7 and 8), the coding sequence is from nucleotides 62-1249. The signal sequence extends from approximately Methyl to Gly24. Thus, "the mature polypeptide extends from about Gln25 to Val395. The chemokine domain extends from about Gln25 to Gly1OO, the stem region extends from about Gly1ol to Gln339, the transmembrane domain extends from about Ala340 to Phe358. and the cytoplasmic domain extends from approximately Ala359 to Val395.The CX3Cine proteins of this invention are defined in part by their physicochemical and biological properties.The biological properties of the CX3 human and mouse cines described herein, for example, CX3Cine Human and CX3 mouse cultures are defined by their amino acid sequence and size of maturity.These must also share the biological properties.CX3 human and mouse molecules exhibit identity at approximately __70-80% amino acids if the signal or mature sequences are compared An expert in the art will be aware of ease that some variations of the sequence can be tolerated, for example, conservative substitutions or remote positions from the helical structures without significantly modifying the biological activity of the molecule. Table 1 shows a sequence alignment of the amino acid sequence CX3 Human (CX3C) with Groa (Gro) of chemokine C-X-C, lymphotactin (Ltn) of chemokine C, and the inflammatory protein of the macrophage lß (MlP-lß) of chemokines C-C.

TABLE 1 Comparison of various chemokines Exon 1 Gro (SEQ ID NO 9) MIPATRSLLCAALLLLATSRLATG LTn (SEQ ID NO.10) MRLLLLTFLGVCCLTPMW "MlP-lβ (SEQ ID NO.11) MKLCVSALSLLLLVAAFCAPGFS CX3 (SEQ ID NO.2) MAPISLSWLLRLATFSHLTVLLAG Exon 2 Gro APIANELRCQCLQTNA. GIHLKNIQSLKVLPSGPHCTQT LTn EGVGTEVLEESSCVNLQTQRLPVQKIKTYII EG AMR MIP APMGSDPPTSCCFSYTARKLPRNFWDYYPTSSL..CSQP CX3 QHHGVTKCNITC. SKMTSKIPVALLIHYQQNQAS..CGKR Exon 3 GRO EVIATLKNGREACLDPEAPLVQKIVQKMLKGVPK LTN AVIFV KRGLKIC ^ UPE-? LKWVLAAIKTVDGRASTRKNMAETVPGTGAQRSTSTAITLTG MIP AWFQTKRSKQVCADPSES VQEYVYDLELN CX3 AIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNG ...

CX3Cucines are present in specific tissue types, for example, neural tissues and the interaction of the protein with a receptor will be important to mediate various aspects of physiology or cell development. Cell types that express message encoding CX3Ccinas suggest that important signals in cell differentiation and development are mediated by them. See, for example, Gilbert (1991) Developmental Biology (3d ed.) Sinauer Associates, Sunderland, MA; Brodwer, et al. (1991) Developmental Biology (3d ed.) Saunders, Philadelphia, PA.; Russo, et al. (1992) Development: The Molecular Genetic Approach Springer-Verlag, New York, N.Y .; and Wilkins (1993) Genetic Analysis of Animal Development (2d ed.) Wiley-Liss, New York, N.Y. In addition, the expression of CX3Ccina must serve to define certain subpopulations of cells.

The CX3C chemokine that produces the profile of various cells is explained here. Screening of a cDNA library generated from brain provides a novel cytokine called CX3 human Cina. The CX3 human Ccinna shows distant similarities with the members of the families of the cytokines C, C-C and C-X-C, with another hitherto unknown number of amino acid residues separating the characteristic cysteines in the motif which is peculiar to and partially defines the chemokines. These observations suggest that CX3Cinas represent the novel additions to the chemokine superfamily. The biochemistry of the CX3C chemokine protein was assessed in mammalian expression systems. Human embryonic kidney 293 cells (HEK293) transfected with mammalian expression constructs encoding full-length CX3C chemokines were metabolically labeled with 35S cysteine and methionine. The CX3C chemokine was produced as a Mr ~ 95kDa protein; the supernatants of the transfected control did not contain these species. Neuraminidase and glucosidases reduced the Mr of the CX3C chemokine from ~ 95kDA to ~ 45kDa, suggesting that the recombinant form is substantially glycosylated. The CX3C chemokine cDNA encoding a predicted membrane bound protein encodes a glycoprotein that is released from the cells by an undefined mechanism.

The pro-migratory activities of CX3C chemokine have been evaluated in icotaqui iotaxis trials. CX3C chemokine appears to be a potent attractant of peripheral blood and T cell monocytes. Pro-migratory activity for neutrophils in the blood has been difficult to demonstrate. The CX3C chemokine gene has been mapped to human chromosome 16. Mapping studies also indicate the possibility of a pseudo gene or related gene on human chromosome 14. Sequencing of the genomic DNA fragments suggests that the chemokine gene CX3C has an intron that begins near or in the codon encoding Ile64. Other intron / exon contours have yet to be mapped, but this will be easily achieved by standard methods. The membrane binding form of CX3Ccina possesses pro-adherent properties for circulating T cells and monocytes. A secreted or soluble form, consisting of the chemokine domain and the stem region, can inhibit this proadhesive activity. This suggests that the membrane binding form of CX3Cycin can be a potent regulator of circulating leukocytes and, in this way, it may be involved in the various inflammatory diseases, for example, arthritis. The soluble form can be used as a pro-adhesion regulator, especially under conditions of compromised immune response. The properties of chemokine CX3C as a chemoattractant of T cells and monocytes, coupled with their distribution in the brain and other organs, suggest that the CX3C chemokine may be involved in the pathogenesis of these inflammatory CNS disorders such as multiple sclerosis and other pathologies that includes neurogenic inflammation. Given that the distribution of CX3C chemokine is not limited to the brain, however, the entire spectrum of inflammatory and immunoregulatory conditions and infections considered as influencing other chemokines should now also be considered for the CX3C chemokine. See, for example, Frank, et al. . { Eds. ) (1995) Samter 's Immunologic Diseases 5a. ed., Vols. I and II, Little, Brown, and Co., Boston, MA.

II. Definitions the term "linkage composition" refers to molecules that bind with specificity to a CX3Cine, for example in an antibody-antigen interaction. However, other compounds, for example, receptor proteins, can also be specifically associated with CX3Cinases for the exclusion of other molecules. Typically, the association will be in a physiologically relevant, covalent or non-covalent natural protein-protein interaction, and may include members of a multiprotein complex, including carrier compounds, or participate in dimerization. The molecule can be a polymer or chemical reagent. No implication, such as if a CX3Cine is the ligand or the receptor of a ligand-receptor interaction, is necessarily presented, unlike if the interaction has similar specificity, for example, specific affinity. A functional analog can be a ligand with structural modifications or it can be a completely unrelated molecule, for example, having a molecular form that interacts with the appropriate determinants that bind to the ligand. Ligands can serve as an agonist or receptor antagonists, see, for example, Goodman et al. (eds.) (1990) Goodman & Gilman's: The Pharmacological Bases of Therapeutics (8th ed.) Pergamon Press, Terrytown, N.Y. The term "protein binding agent complex: CX3Cine" is used herein to refer to a complex of a binding agent and a CX3Cine protein that is formed by the specific binding of the binding agent to the CX3Cine protein, for example, preferably the domain chemokine The specific binding of the binding agent means that the binding agent has a specific binding site that recognizes on site in the CX3Cine protein. for example, the antibodies developed for a CX3Cine protein and that recognize an epitope in the CX3Cine protein are capable of forming a complex binding agent: CX3Cine protein by specific binding. Commonly, the formation of a complex binding agent: CX3Cine protein allows the measurement of a CX3Cine protein in a mixture of other proteins and biological substances. The term "antibody complex: CXSCin protein" refers to an embodiment in which the binding agent is an antibody. The antibody can be monoclonal, polyclonal or a binding fragment of an antibody, for example, a Fab fragment of F (ab2). The antibody will preferably be a polyclonal antibody for purposes of cross-reactivity. The "homologous" nucleic acid sequences, when compared, show significant similarity. Standards for homology in nucleic acids are measures for homology generally used in the art by comparison of the sequence and / or phylogenetic relatiop, or based on the conditions of hybridization. The conditions of the hybridization are described in more detail below. An "isolated" nucleic acid is a nucleic acid, for example, an RNA, DNA or a mixed polymer which is substantially separated from other biological components which naturally accompany a native sequence, eg, proteins and genomic sequences of flanking from the originating species. The term comprises a nucleic acid sequence that has been removed in its natural environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or biologically analogs synthesized by heterologous systems. A substantially pure molecule includes isolated forms of the molecule. An isolated nucleic acid will usually contain homogeneous nucleic acid molecules, but in some embodiments it will contain nucleic acids with less sequence heterogeneity. This heterogeneity is usually found at polymer ends or non-critical portions for a desired biological function or activity. As used in this, the term "CX3Cine protein" should comprise, when used in the context of a protein, a protein having an amino acid sequence, particularly from the chemokine moieties, as shown in SEQ ID NO: 2, 4, 6 or 8, or a significant fragment of this protein, for example, preferably the chemokine domain. The invention also comprises a polypeptide having a similar structure to CX3, either human or mouse, for example, which interacts with specific binding components of CX3Cine. These linkage components, for example, antibodies, usually bind to a CX3Cine with higher affinity, for example at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM and higher preference better than about 3 nM. The term "polypeptide" or "protein" as used herein includes a significant fragment or segment of the chemokine moiety portion of a CX3Cine, and comprises an amino acid residue elongation of at least about 8 amino acids, generally at least 10 amino acids, more generally at least 12 amino acids, often at least 14 amino acids, much more frequently at least 16 amino acids, usually at least 18 amino acids, more commonly at least 20 amino acids, normally at least 22 amino acids, more usually at least 24 amino acids, preferably at least 26 amino acids, more preferably at least 28 amino acids and in the particularly preferred embodiments at least about 30 or more amino acids, for example, 35, 40, 45 , 50, 60, 70, 80, etcetera.

A recombinant nucleic acid is defined by its production method or structure. In reference to this production method, for example, a product made by a process, the process makes use of recombinant nucleic acid techniques, for example, it includes human intervention in the nucleotide sequence, usually the selection or production in an alternative way, it can be an elaborated nucleic acid generating a sequence comprising the fusion of two fragments that in their natural state are not contiguous with each other, but it is tried to exclude products of nature, for example, mutants that appear in natural. Thus, for example, products made by transforming cells with any vector that is not found naturally are understood, as they are nucleic acids containing sequence derived using any synthetic process of oligonucleotides. This is often done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while a sequence recognition site is usually introduced or removed. Otherwise, it is performed to join together the nucleic acid segments of desired functions to generate a single genetic entity that contains a combination of desired functions that are not found in the commonly available natural forms. Recognition sites for restriction enzymes are often the target of these artificial manipulations, but other site-specific targets, for example, promoters, DNA replication sites, regulatory sequences, control sequences or other useful features can be incorporate by design. A similar concept is proposed for a recombinant, for example, fusion, polypeptide. Specifically included are synthetic nucleic acids, which, by redundancy of the genetic code, encode polypeptides similar to fragments of these antigens, and fusions of sequences from various variants of different species. Mutations of the protease cleavage sites can also be carried out. "Solubility" is reflected by sedimentation measured in Svedberg units, which are a measure of the sedimentation rate of a molecule under specific conditions. The determination of sedimentation velocity was traditionally performed in an analytical ultracentrifuge, but currently it is usually done in a normal ultracentrifuge. See, Freifelder (1982) Physical Biochemistry (2nd ed.).

H. Freeman & Co., San Francisco, CA; and Cantor and Schimmel (1980) Biophyscal Chemistry, parts 1-3,. H. Freeman & Co., San Francisco CA. As a crude determination, a sample containing a putatively soluble polypeptide is centrifuged in a normal size ultracentrifuge at approximately 50K rpm for about 10 minutes, and the soluble molecules will remain in the supernatant. A soluble particle or polypeptide will usually be less than about 30S, more commonly, less than about 15S, usually less than about IOS, more commonly less than about 6S, and, in particular embodiments, preferably less than about 4S. and more preferably less than about 3S. The solubility of a polypeptide or fragment will depend on the medium and the polypeptide. Many parameters affect the solubility of the polypeptide, including temperature, the electrolyte medium, size and molecular characteristics of polypeptide and nature of the solvent. Typically, the temperature of which the polypeptide is used is in the range of about 4 ° C to about 65 ° C. Typically, the temperature in use is greater than about 18 ° C and more commonly greater than about 22 ° C. For diagnostic purposes, the temperature will usually be about room temperature or warmer but less than the denaturing temperature of the components in the test. For therapeutic purposes, the temperature will usually be body temperature, usually around 37 ° C for humans, although in certain situations the temperature can be elevated or lowered in situ or in vitro. The size and structure of the polypeptide in general should be evaluated in a substantially stable state, usually not in a denatured state. The polypeptide may be associated with other polypeptides in a quaternary structure, for example, to confer solubility or associated with lipids or detergents in a manner that approximates the natural interactions of the lipid bilayer. The solvent will usually be a biologically compatible buffer solution of a type used to preserve biological activities, and will usually approximate a physiological solvent. Usually, the solvent will have a neutral pH, usually between about 5 and 10, and preferably about 7.5. Sometimes a detergent will be added, usually a moderate non-denaturing, for example, CHS (cholesteryl emisuccinate) or CHAPS (3- [3-colamidopropyl (dimethylammonium-1-propane) sulfonate, or a rather low concentration as to avoid significant breakdown of the structural or physiological properties of the protein. "Substantially pure" in the context of a protein usually means that the protein is isolated from other contaminating proteins, nucleic acids and other biological substances from the original strong organism The purity or "isolation" can be tested by normal methods, and will usually be at least about 50% pure, more regularly at least about 6CT% pure, generally at least about 70% pure, more generally at least about 80% pure, often at least about 90% pure, preferably at least about 95% more preferably at least about 98% pure and in the most preferred embodiments at least 99% pure. Similar concepts apply, for example, to antibodies or nucleic acids. "Substantial similarity" in the context of comparing the nucleic acid sequence means that the segments, or their complementary strands, when compared are identical when they are aligned optimally, with insertions or exhaustions of suitable nucleotides, at least about 50% of the nucleotides, generally at least 56%, more generally at least 59%, usually at least 62%, more commonly at least 65%, often at least 68%, with greater frequency at least 71%, usually at least 74%, more regularly at least 77%, usually at least 80%, most commonly at least about 85%, preferably at least about 90% , more preferably at least about 95 to 98% or more, and in specific embodiments, as high as about 99% or more of the nucleotides. Otherwise, the substantial similarity exists when the segments will hybridize under selective hybridization conditions, up to one strand or its complement, usually using a sequence from SEQ ID NO: 1, 3, 5 or 7. Usually, the Selective hybridization will occur when there is at least about 55% similarity over an elongation of at least about 30 nucleotides, preferably at least about 65% over an elongation of at least about 25 nucleotides, more preferably at least about 75%, and most preferably at least about 90%, about 20 nucleotides. See Kanehisa (1984) Nuc. Acids Res. 12: 203-213. The length of the similarity comparison, as described, may be on larger elongations, and in certain embodiments it will be on an elongation of at least 17 nicleotides, usually at least about 20 nucleotides, more commonly at least about 24 nucleotides, usually at least about 28 nucleotides more regularly at least about 40 nucleotides, preferably at least about 50 nucleotides, and most preferably at least about 75 nucleotides to 100 nucleotides; more nucleotides, for example, 150, 200, et cetera. - "Severe conditions", in relation to the homology or substantial similarity in the context of hybridization, will be in severe conditions, combined salt, temperature, organic solvents and other parameters, usually those that are controlled in hybridization reactions. The combination of the parameters is more important than the measurement of any of the individual parameters. Severe temperature conditions will usually include temperatures in excess of about 30 ° C, more commonly in excess of about 37 ° C, by regulating in excess of about 45 ° C, more regularly in excess of about 55 ° C, preferably in excess of about 65 ° C, and more preferably in excess of about 70 ° C. Severe salt conditions will usually be less than about 1000 mM, usually less than about 500 mM, more common less than about 400 mM, to regulate less than about 300 mM, preferably less than about 200 mM, and greater preference less than about 150 mM, see, for example, et ur and Davidson (1968) J. Mol. Biol. 31: 349-370. A nucleic acid probe that binds to a target nucleic acid under severe conditions is specific for this target nucleic acid. This probe is usually more than 11 nucleotides in length and is sufficiently identical or complementary to the target nucleic acid over the region specified by the probe sequence to bind the target under severe hybridization conditions. The CX3Ccinas from other mammalian species can be cloned and isolated by cross-hybridization of closely related species. See, for example, below. The similarity may be relatively low between distinctly related species, and thus the hybridization of closely related species in a relet form is advisable. Otherwise, the preparation of an antibody preparation that exhibits less specificity to the species may be useful in expression cloning methods. The phrase "binds specifically to an antibody" or "specifically immunoreactive with", when referring to a protein or peptide refers to a binding reaction that is determinative in the presence of the protein in the presence of a heterogeneous population of proteins and other biological components. In this way, under the name of immunoassay conditions, the specific antibodies bind to a specific protein and do not bind significantly to other proteins present in the sample. Specific binding to an antibody under these conditions may require that an antibody be selected for its specificity for a specific protein. For example, the antibodies developed for the CX3 protein immunogen Human with the amino acid sequence represented in SEQ ID NOs 2, 4, 6, or 8 can be selected to obtain antibodies specifically immunoreactive with CX3 proteins and not with other proteins. . These antibodies recognize proteins that are highly similar to CX3 proteins.

III. Nucleic Acids CX3 Human Cina is an example of a larger class of structurally and functionally related proteins. These soluble chemokine proteins will serve to transmit signals between different cell types. Preferred embodiments, as described, will be useful in normal procedures for isolating genes from different individuals or other species, for example, warm-blooded animals, such as birds and mammals. Cross-hybridization will allow the isolation of related genes that encode proteins from individuals, strains or species. Various different methods are available to successfully isolate a suitable nucleic acid clone based on the information proposed herein. Southern blot hybridization studies can qualitatively determine the presence of homologous genes in human, monkey, rat, dog, cattle and rabbit genomes under specific hybridization conditions. The complementary sequences will also be used as probes or primers. Based on the identification of the most suitable amino terminus, other peptides should be particularly useful, for example, coupled with the techniques of the anchored vector or poly-a complementary PCR c with DNA complementary to other peptides. In addition, reverse translation using redundancy in the genetic code can provide synthetic genes that can encode essentially identical proteins frequently with a selection of the preferred codon usage for expression in a given host cell. Techniques for the manipulation of the nucleic acid of the genes encoding CX3Ccina proteins, such as subcloning nucleic acid sequences encoding polypeptides in expression vectors, marker probes, DNA hybridization and the like, are generally described in Sambrook, et al. to the. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, which is incorporated herein by reference. This manual will be referred to below as "Sambrook, et al." - There are several methods of isolation of :, DNA sequences that encode CX3Ccina proteins. For example, the DNA is isolated from a genomic or cDNA library using labeled probes of oligonucleotides having sequence identical or complementary to the sequences described herein. It is possible to use full-length probes or probes. of oligonucleotides can be generated by comparison of the described sequences. These probes can be used directly in hybridization assays to isolate DNA encoding CX3Ccina proteins, or it is possible to design primers, for example, using the flanking sequence, for use in amplification techniques such as PCR, for the isolation of DNA that encodes the CX3Ccinas proteins. To prepare a cDNA library, the mRNA is isolated from cells that express a CX3 protein. The cDNA is prepared from the mRNA and ligated into a recombinant vector. The vector is transfected in a recombinant host for its propagation, screening and cloning. The methods for making and screening cDNA libraries are well known. See Gubler and Hoff an (1983) Gene 25: 263-269 and Sambrook, et al.

For a genomic library the DNA can be extracted from tissue and can be mechanically cut or enzymatically digested to produce fragments, for example, of approximately 12-20 kb. The fragments are then separated by gradient centrifugation and cloned in bacteriophage landa vectors. These vectors and phages are packed in vitro, as described in Sambrook et al. The recombinant phage is analyzed by plaque hybridization as described in Benton and Davis (1977) Science 196: 180-182. Colony hybridization is brought to v_abo as generally described in, for example, Grunstein et al. (1975) Proc. Nati Acad. Sci. USA. 72: 3961-3965. The DNA encoding a CX3Cine protein can be identified in cDNA or genomic libraries for its ability to hybridize with r.ucleic acid probes described herein, for example, in colony or plate hybridization assays. The corresponding DNA regions are isolated by standard methods known to those skilled in the art. See, for example, Sambrook, et al. Otherwise, the sequence databases, for example, GenBank, can be evaluated for similar or corresponding sequences. Different methods of amplification of target sequences, such as the polymerase chain reaction, can also be used to prepare DNA that encodes proteins CX3Ccina. Polymerase chain reaction (PCR) technology is used to amplify these nucleic acid sequences directly from mRNA, cDNA and from genomic libraries or cDNA libraries. The isolated sequences that encode CX3Ccina protein can also be used as templates for PCR amplification. Typically, PCR techniques synthesize complementary oligonucleotide primers for two 5f regions in two strands of the region of the DNA to be amplified. The polymerase chain reaction is then carried out using the two opposite primers. See, Innis, et al. (eds.) (1990) PCR Protocols: A Guide to Methods and Applications Academic Press, San Diego, CA. The primers can be selected to amplify whole regions that encode a full-length CX3 protein or to amplify smaller DNA segments, as desired. Once these regions are amplified by PCR; these can be sequenced and the oligonucleotide probes can be prepared from the sequence obtained using standard techniques. These probes can then be used to isolate the DNAs that encode the CX3Cucins proteins. Oligonucleotides for use as probes are usually chemically synthesized according to the solid phase triester phosphoramidite method described for the first time by Beacage and Carruthers (1983) Tetrahedron Lett. 22 (20): 1859-1862, using an automated synthesizer, as described in Needham-VanDevanter, et al. (1984) Nucleic Acids Res. 12: 6159-6168. The purification of the oligonucleotides is carried out, for example, by natural acrylamide gel electrophoresis or by HPLC. anion exchange as described in Pearson and Regnier (1983) J. Chrom. 255: 137-149. The sequence of the synthetic oligonucleotides can be verified using, for example, the chemical degradation method of Maxam, AM and Gilbert, W. In Grossman, L. and Moldave (eds.) (1980) Methods in Enzymology 65: 499-560 Academic Press, New York. An isolated nucleic acid encoding a human CX3 protein was identified. The nucleotide sequence and the corresponding open reading frame are provided in SEQ ID NOs 1 and 2; with other sequences that are provided in SEQ ID NOs 3 and 4. Correspondingly, a mouse sequence was identified and its corresponding open reading frame and nucleotide are provided as SEQ ID NOs: 5-8. These CX3Cinas have limited similarity with portions of chemokines, specifically the chemokine domains. See, for example, Matssushima Oppenheim (1989) Cytokine 1: 2-13; Oppenheim, et al. (1991) Ann. Rev. Immunol. 9: 617-648; Schall (1991) Cytokine 3: 165-183; and Gronenborn and Clore (1991) Protein Engineering 4: 263-269. In particular, CX3 human shows similarity to class C chemokines in the carboxyl terminal portion, specifically with respect to the length and corresponding positions, in the numbering of the mature human sequence, to the sequence cys-ala-asp-pro in positions 50-53; and the prp-val sequence at positions 57-58. CX3Cinas have a carboxyl terminal tail much longer than members of the CC or CXC chemokine families, and this "stem" region may play a role in the presentation of chemokine. It should be noted that the separation of the cysteine residues conserved in the CXC and CC families of the chemokines are absent in the CX3 human modality. Other characteristics of the comparison are evident between the CX3Ccina families and chemokines. See, for example, Lodi, et al. (1994) Science 263: 1762-1766. In particular, ß-sheet and a-helix residues can be determined, using for example the RASMOL program, see Sayle and Milner-White (1995) TIBS 20: 374-376; or Gronenberg, et al. (1991) Protein Enginnering 4: 263-269; and other structural characteristics are defined in Lodi, et al. (1994) Science 263: 1762-1767.

These secondary and tertiary characteristics help to define more of the structural characteristics of C, CC and CXC, together with the spacing of the appropriate cysteine residues. - Based on structural modeling and perceptions in the binding regions of chemokines, it can be predicted that the residues in the mature human protein, lacking a 24 residue signal, 26 (his), 28 (gln), 40 (ile), 42 (glu), 47 (arg) and 48 (leu) must be important for the binding of chemokines to cells. The residues in the amino terminus are probably not involved in specificity or receptor binding. In addition, the exon boundaries are predicted to correspond to the segments of the protein that include the signal sequence through approximately the second residue (his) in the mature protein; from there to approximately three residues after the third cys (around arg-ala); and from there to the end. The third exon appears to have relatively high similarity with the other chemokines. The second exon would probably be more characteristic of the CX3C chemokines, and would be the preferred segment to be used to investigate homology in other variants, eg, species or others. In particular, the segments that are expected to be preferred in the production of CX3C-specific chemokine antibodies will include peptides or sequences in the region from the second residue of the mature protein (his) to approximately the third residue after the third cysteine (arg). . Fragments of at least about 8-10 residues in this region would be especially interesting peptides, for example, starting at the residue positions of 1,2,3 etc., mature. These fragments would usually end up in this region, for example, in residue 37, 36, 35, etc. Other interesting peptides of various lengths would include those that start or end at other positions of the protein, for example, at residues 87, 86, etc., with lengths in the range of, for example, about 8 to 20, 25, 30 , 35, 40, etc. Corresponding fragments of other CX3 mammalian, eg mouse, chambers would be the preferred embodiments. This invention provides isolated DNA or fragments for encoding a CX3Cycin protein. In addition, this invention provides isolated or recombinant DNA encoding a protein or polypeptide that is capable of hybridizing under suitable conditions, e.g., high stringency, to the DNA sequences described herein. The biologically active protein or polypeptide can be an intact ligand, or fragment, and have an amino acid sequence as described in SEQ ID NO 2, 4, 6, or 8. Preferred embodiments will be full length natural sequences, from isolates, for example from about 11,000 to 12,500 daltons in size when they are not glycosylated or fragments of about 6,000 daltons, most preferably at least about 8,000 daltons In the glycosylated form, the protein can exceed 12,500 daltons. In addition, this invention contemplates the use of isolated or recombinant DNA, fragments of this, which encodes proteins that are homologous to a CX3 protein or that were isolated using cDNA encoding a CX3 protein as a probe. The isolated DNA can have the respective regulatory sequences on the 5 'and 3' flanks, for example, promoters, enhancers, poly-a addition signals and others.

IV. Preparation of CX3Cine The DNAs encoding a CX3Cine or fragments thereof can be obtained by chemical synthesis, screening of cDNA libraries or screening of genomic libraries prepared from a wide variety of cell lines or tissue samples. Genetic provides a variety of polynucleotide sequences that must encode the same protein.These DNAs can be expressed in a wide variety of host cells for the synthesis of a full-length protein or fragments that, in turn, for example, can be used to generate polyclonal or monoclonal antibodies, for binding studies, for the construction and expression of modified molecules, and for structure / function studies.Each CX3Cine or its fragments, for example, the chemokine domain, can be expressed in host cells that are transfected or transformed with suitable expression vectors. they are substantially purified to be free of protein or cellular contaminants other than those derived from the recombinant host, and are therefore particularly useful in pharmaceutical compositions when combined with a pharmaceutically acceptable carrier and / or diluent. The antigen, for example, CX3Coin or portions thereof, may be expressed as fusions with other proteins or may possess an epitope tag. This also applies to antigen binding sites. The expression vectors by io common are self-replicating DNA or RNA constructs containing the desired antigen gene or its fragments, usually operably linked to the appropriate genetic control elements that are recognized in a suitable host cell. The specific type of control elements necessary to effect the expression will depend on the host cell that is used. Typically, the elements of genetic control may include a prokaryotic promoter system or a control system of eukaryotic promoter expression, and will usually include a transcription promoter, an optional operator to control the initiation of transcription, enhancers of the transcription to raise the level of mRNA expression, a sequence encoding an appropriate ribosome binding site, and sequences that complete transcription and translation. Expression vectors also usually contain an origin of replication that allows the vector to replicate independently of the host cell. The vectors of this invention comprise the DNAs encoding a CX3Ccina, or a fragment thereof, usually encoding, for example, a biologically active polypeptide or protein. The DNA can be under the control of a viral promoter and can encode a selection marker. This invention further contemplates the use of these expression vectors that are capable of expressing eukaryotic cDNA encoding a CX3Cine protein in a prokaryotic or eukaryotic host, wherein the vector is compatible with the host, and wherein the eukaryotic cDNA encoding the protein is inserted into the vector so that the growth of the host containing the vector expresses the cDNA in question. Typically, expression vectors are designed for stable replication in their host cells or for amplification to greatly increase the total number of copies of the desirable gene per cell. It is not always required that an expression vector replicate in a host cell, for example, it is possible to effect the transient expression of the protein or its fragments in various hosts using vectors that do not contain an origin of replication that is recognized by the host cell. It is also possible to use vectors that -cause the integration of a CX3Ccina gene or its fragments into the host DNA by recombination, or integrate a promoter that controls the expression of an endogenous gene. The vectors, as used herein, contemplate plasmids, viruses, baceteriofagos, integrable DNA fragments and other vehicles that allow the integration of DNA fragments in the host genome. Expression vectors are specialized vectors that contain elements of genetic control that effect the expression of linked genes operably. Plasmids are the vector form that is used most frequently, but many other forms of vectors that perform an equivalent function are suitable for use herein. See, for example, Pouwels, et al. (1985 and Supplements) Cloning Vectors: A Laboratory Manual Elsevier, N.Y.; and Rodriguez, et al. (eds.) (1988) Vectors: A Survey of Molecular Clonin Vectors and Their Uses Butterworth, Boston, MA.

Suitable host cells include prokaryotes, lower eukaryotes and higher eukaryotes. Prokaryotes include gram negative and gram positive organisms, for example, E. coli and B. subtilis. Lower eukaryotes include yeasts, for example, S. cerevisiae and Pichia, and species of the genus Dictyostelium. Higher eukaryotes include cell lines of established tissue cultures from animal cells, both of non-mammalian origin, eg, insect cells, and birds and of mammalian origin, eg, humans, primates and rodents. The pro-biotic-vector host system includes a wide variety of vectors for very different species. As used herein, E. coli and its vectors will be used generically to include equivalent vectors that are used in other prokaryote =. A representative vector for amplifying DNA is pBR322 or its derivatives. Vectors that can be used to express CX3Ccinas' or fragments of CX3Cucines include, but are not limited to, vectors such as those containing the lac promoter (from the pUC series); trp promoter (pBR322-trp); Ipp promoter (the pIN series); lambda-pP or pR promoters (pOTS); or hybrid promoters such as ptac (pDR540). See Brosius, et al. (1988) "Expression Vectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters", in Rodríguez and Denhardt (eds.) Vectors: A Survey of Molecular Cloning Vectors and Their Uses 10: 205-236 Buttersworth, Boston , MA. Lower eukaryotes, for example, yeast and Dictyostelium can be transformed with vectors containing the sequence of CX3Cine. For purposes of this invention, the most common lower eukaryotic host is the baking yeast, Saccharomyces cerevisiae. This will be used in a generic way to present the lower eukaryotes, although other strains and species are also available. Yeast vectors usually consist of an origin of replication (except of the integrating type), a selection gene, a promoter, DNA encoding the desired protein or its fragments and sequences for translation termination, polyadelination and termination of transcription. Suitable expression vectors for yeast include constitutive promoters such as 3-phosphoglyceratokinase and some other promoters of the glycolytic enzyme gene or inducible promoters such as the alcohol dehydrogenase 2 promoter or metallothionine promoter. Suitable vectors include derivatives of the following types: auto replicant copy number (such as the YRp series), self-replicating high copy number (such as the YEp series); integral types (such as the YIp series); or mini-chromosome (like the YCp series).

Higher eukaryotic tissue culture cells are usually the preferred host cells for the functional CX3 active protein expo-ion. In principle, it is possible to use a large number of higher eukaryotic tissue culture cell lines, for example, insect baculovirus expression systems, either from an invertebrate or vertebrate source. However, mammalian cells are preferred to carry out the appropriate processing, both for co-translation and post-translation. The transformation or transfection and propagation of these cells is routine. Useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, newborn rat kidney cell lines (BRK), insect cell lines, bird cell lines and cell lines. of monkey (COS). Expression vectors for these cell lines usually include an origin of replication, a promoter, a translation initiation site, RNA binding sites (eg, if genomic DNA is used), a polyadenylation site, and a transcription termination site. These vectors may also contain a selection gene or amplification gene. Suitable expression vectors can be plasmids, viruses or retroviruses carrying promoters from, for example, sources such as adenovirus, SV40, parvovirus, vaccinia virus or cytomegalovirus. Representative examples of suitable expression vectors include pCDNAl; pCD; see Okamaya, et al. (1985) Mol. Cell Biol. 5: 1136-1142; Poly-A polymerase, see Thomas, et al. (1987) Cell 51: 503-512; and a baculovirus vector such as pAC 373 or pAC 610. It is very likely that CX3Cucines do not need to be glycosylated to manifest biological responses. However, sometimes it will be desirable to express a CX3Cycin polypeptide in a system that provides a specific or defined glycosylation pattern. In this case, the normal pattern will be that provided in a natural way by the expression system. However, the pattern will be odifiable by exposing the polypeptide, for example, in non-glycosylated form, to the appropriate glycosylating proteins introduced in a heterologous expression system. For example, the CX3Ccina gene can be co-transformed with one or more genes that encode mammalian enzymes or other glycosylating enzymes. Furthermore, it is understood that over glycosylation can cause damage to the biological activity of CX3Ccina, and that the expert can perform routine tests to optimize the degree of glycosylation that confers optimal biological activity. A CX3Ccina, or a fragment thereof, can be designed to be phosphatidyl inositol (Pl) bound to a cell membrane, but can be removed from the membranes by treatment with an overdose of phosphatidyl inositol, for example, phosphatidyl inositol phospholipase- C. It releases the antigen in a biologically active form and allows purification by the normal procedures of protein chemistry. See, for example, Low (1989) Biochem. Biohys. Acta 988: 427-454; Tse, et al. (1985) Science 230: 1003-1008; and Brunner, et al. (1991) J. Cell Biol. 114: 1275-1283. Now that the CX3Kines have been characterized, it is possible to prepare fragments or derivatives thereof by conventional processes to synthesize peptide. These include processes such as those described Stewart and Young (1984) Solid Phase Peptide Synthesis Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis Springer-Verlg, New York, NY; and Bodanszky (1984) The Principies of Peptide Synthesis Springer-Verlg, New York NY. For example, an azide process, a process with hydrochloric acid, a process with acid anhydride, a process with combined anhydride, an active ester process (for example, p-nitrophenylester, N-hydroxysuccinimide ester, or cyanomethyl ester), a carbodiimidazole process , an oxidative-reductive process or a dicyclohexycarbodiimide (DCCD) / additive process can be used. The solid phase and solution phase syntheses are applicable to the aforementioned processes. The protein and the fragments of these preparations can be isolated and purified from the reaction mixture by means of separation of peptides, for example, by extraction, precipitation, electrophoresis and various forms of chromatography, and the like. The CX3Cines of this invention can be obtained in varying degrees of purity depending on their intended use. The purification can be carried out by the use of known protein purification techniques or by the use of the antibodies or against binding sites described herein, for example, in immunoabsorbent affinity chromatography. See, for example, Coligan, et al. (eds.) (1995 and Periodic Supplements) Current Protocols in Protein Science, John Wiley and Sons, New York, NY. This immunoabsorbent affinity chromatography is carried out by first attaching the antibodies to a solid support and then contacting the solubilized-linked antibodies with the appropriate source cells, used from other cells that express the ligand or used or supernatants of cells that produce the CX3Kitches as a result of recombinant DNA techniques, see below.

The multiple cell lines can be screened for one that expresses a CX3Ccina at a high level compared to other cells. Various cell lines, for example, a line of stromal cells from mouse thymus TA4, are screened and selected for their favorable handling properties. The natural CX3Cucines can be isolated from natural sources or by expression from a transformed cell using an appropriate expression vector. The purification of the expressed protein is carried out by normal procedures or can be combined with means designed for effective purification at high efficiency from used or cellular supernatants. The epitope or other tags, for example, FLAG or His? Segments, can be used for these purification aspects.

V. Antibodies It is possible to develop antibodies for various CX3Cucines, including individual, polymorphic, allelic variants of strains or species and fragments thereof, both in their forms and occur in nature (full length) or in their recombinant forms. In addition, antibodies can be developed to CX3Ccinas in their active or native forms or in their inactive or denatured forms. It is also possible to use anti-idiotypic antibodies.

A. Production of antibodies A large number of immunogens can be used to produce antibodies specifically reactive with CX3 protein. The recombinant protein is a preferred immunogen for the production of monoclonal or polyclonal antibodies. The protein found in nature can also be used in its pure or impure form. Synthetic peptides, prepared using the CX3 protein sequences of human or rerum described herein, can also be used as an immunogen for the production of antibodies to CX3Cycines, for example, the chemokine domains thereof. The recombinant protein can be expressed in eukaryotic or prokaryotic cells as described herein and can be purified as described. It is possible to use bent material in natural or denatured form, as appropriate, to produce antibodies. Monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassay to measure the protein. Methods of producing polyclonal antibodies are known to those skilled in the art. Usually, an immunogen, preferably a purified protein, is mixed with an adjuvant and the animals are immunized with the mixture. The immune response of the animals to the immunogen preparation is verified by taking blood samples and determining the titre of the reactivity to the CX3 protein or fragment of interest. When suitably elevated titers of the antibody are obtained for the immunogen, usually after repeated immunizations, blood is collected from the animal and the antisera are prepared. If desired, it is possible to carry out another fractionation of the antisera to enrich the antibodies reactive to the protein. See, for example, Harlow and Lane; or Coligan. Monoclonal antibodies can be obtained by different techniques known to those skilled in the art. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, usually by fusion with a myeloma cell (see, Kohler and Milstein (1976) Eur. J. Immunol. 6: 511-519, "which is incorporated herein by reference.) Alternative methods of immortalization include transformation with the Epstein Barr virus, oncogenes or retroviruses or other methods known in the art." Colonies that develop from individual immortalized cells are screened for the production of antibodies of specificity and affinity desired for the antigen, and the yield of the monoclonal antibodies produced by these cells can be improved by various techniques, including injection into the peritoneal cavity of a vertebrate host, otherwise it is possible to isolate DNA sequences that encode a monoclonal antibody or a binding fragment that is screening a library of DNA from human B cells according to, for example, the general protocol mentioned by Huse, et al. (1989) Science 246: 1275-1281. Antibodies, including binding fragments and individual strand versions, against the predetermined fragments of CX3Kines can be developed by immunization in animals with conjugates of the fragments with carrier proteins as described above. The monoclonal antibodies are prepared from cells that secrete the desired antibody. These antibodies can be screened for binding to CX3 Normal or defective carcasses, or they can be screened for agonist or antagonist activity, for example mediated by a receptor. These monoclonal antibodies will usually bind with at least one KD of about 1 mM, more commonly at least about 300 μM, usually at least about 10 uM, more regularly at least about 30 μM, preferably by at least about 10 μM, and most preferably at least about 3 μM, or better. - In some cases it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. The description of the techniques for preparing monoclonal antibodies can be found in, for example, Stites, et al. (eds.) Basic and Clinical Immunoiogy (4th ed.) Lange Medical Publications, Los Altos CA, and references mentioned therein; Harlow and La e (1988) Antibodies: A Laboratory Manual CSH Press; Goding (1986) Monoclonal Antibodies: Principies and Practice (2nd ed.) Academic Press, New York, NY; and in particular in Kohler and Misltein (1975) Nature 256: 495-497, which describe a method for generating monoclonal antibodies. In short, this method includes injecting an animal with an immunogen. The animal is then sacrificed and the cells in its vessel are taken, which are then fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secretes a single species of antibody to the immunogen. In this form, the individual antibody species that are obtained are the individual B cell products cloned and immortalized from the immune animal generated in response to a specific recognized site on the immunogenic substance. Other suitable techniques include the selection of antibody libraries in phages or similar vectors. See, for example, Huse, et al. (1989) "Generation of Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambadat ~" Science 246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546. The polypeptides and antibodies of the present invention can be used with or without modification, including chimeric or humanized antibodies. Frequently the polypeptides and antibodies will be labeled by binding, covalently or non-covalently, a substance that provides a detectable signal. A wide variety of brands and conjugation techniques are known and widely reported in the scientific and patent literature. Suitable labels include radionuclides, enzymes, cofactor substrates, inhibitors, fluorescent moieties, chemiluminescent portions, magnetic particles, and the like. Patents showing the use of these trademarks include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Likewise, it is possible to produce recombinant immunoglobulins. See Cabilly, U.S. Patent No. 4,816,567; and Queen, et al. (1989) Proc. Nat'l Acad. Sci. USA 86: 10029-10033.

The antibodies of this invention are useful for chromatography by affinity in the isolation of CX3 protein. It is possible to prepare columns where the antibodies are bound to a solid support, for example, particles, such as agarose, SEPAHDEX or the like in which a lysate or supernatant of cells can be passed through the column, the column is washed, followed by the increase in concentrations of a moderate denaturant, by means of which purified CX3 protein will be released. The antibodies can also be used to screen expression libraries for specific expression products. In general, the antibodies used in this procedure will be marked with a portion that allows easy detection of the presence of antigen by binding to the antibody. Antibodies to CX3Cucines can be used for the identification of populations of cells that express CX3Ccinas. By testing "the expression products by cells expressing CX3Cycines, it is possible to diagnose diseases, for example, states compromised with the immune system.Antibodies that develop against each CX3Cacinas will also be useful to develop anti-idiotypic antibodies. they will be useful to detect or diagnose various immunological states related to the expression of the respective antigens.

B. Immunoassays A specific protein can be measured by various immunoassay methods. For a review of immunological and immunoassay procedures, in general, see Stites and Terr (eds.) (1991) Basic and Clinical Immulogy (7th ed.). In addition, the immunoassays of the present invention can be carried out in various configurations, which is extensively reviewed in Maggio (ed) (1980) Enzyme Immunoassay CRC Press, Boca Raton, Florida; Tijan (1985) "Practice and Theory of Enzyme Immunoassay", Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publisher B.V. Amsterdam; and Harlow and Lane, Antibodies, A Laboratory Manual, supra, each of which is incorporated herein by reference. See also, Chan (ed.) (1987) Immunoassay: A Practical Guide, Academic Press, Orlando, Florida, FL; Price and Newman (eds.) (1991) Principles and Practice of I munoassays Stockton Press, NY; and Ngo (ed.) (1988) Non-isotopic Immunoassays Plenum Press, NY. Immunoassays for the measurement of CX3Cucine proteins can be carried out by various methods known to those skilled in the art. In brief / the immunoassays to measure the protein can be competitive or non-competitive binding assays. In competitive binding assays the sample to be analyzed competes with an analyte labeled by the specific binding sites on a capture agent bound to a solid surface. Preferably the capture agent is an antibody specifically reactive with CX3Cin protein produced as described above. The concentration of the labeled analyte bound to the capture agent is inversely proportional to the amount of free analyte present in the sample. In a competitive binding immunoassay, the CX3Cine protein present in the sample competes with the labeled protein to bind to a specific binding agent. For example, an antibody specifically reactive with the CX3Cycin protein. The binding agent can be attached to a solid surface to effect the separation of the labeled, bound protein from the unbound labeled protein. Otherwise the competitive binding assay can be carried out in the liquid phase and it is possible to use a variety of known techniques to separate the bound labeled protein from the unbound labeled protein. After separation the amount of bound labeled protein is determined. The amount of protein present in the sample is inversely proportional to the amount of binding of the protein.

In an alternative form, it is possible to perform a homogeneous immunoassay in which a separation step is not necessary. In these immunoassays, the mark on the protein is modified by binding the protein to its specific binding agent. This modification in the labeled protein gives rise to a decrease or increase in the signal emitted by the tag, so that the measurement of the tag at the end of the immunoassay allows the detection or quantification of the protein. It is also possible to determine CX3Cycine proteins by a series of non-competitive immunoassay methods. For example, a two-site solid phase sandwich immunoassay can be used. In this type of assay, a binding agent for the protein, for example, an antibody, binds to a solid support. A second binding agent is labeled to the protein, which may also be an antibody, and which binds to the protein at a different site. After binding at both sites of the protein has occurred, the labeled, unbound binding agent is removed and the amount of labeled binding agent that has bound to the solid phase is measured. The amount of bound binding agent bound is directly proportional to the amount of the protein in the sample. It is possible to use Western blot analysis to determine the presence of CX3Cains proteins in a sample. The electrophoresis can be carried out, for example, in a tissue sample suspected of containing the protein. After electrophoresis to separate the proteins and transfer the proteins to a suitable solid support, for example, a nitrocellulose filter, the solid support is incubated with an antibody reactive with the protein. This antibody can be labeled, or otherwise, detected by subsequent incubation with a labeled second antibody that binds to the primary antibody. The immunoassay formats described above employ labeled components for the assay. The label can be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. It can make use of a wide range of brands and methods. A radioactive label incorporating 3H, 125I, 35S, 14C or 32P is usually used. Non-radioactive labels include labeled antibody binding ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies that can serve as specific binding members for a labeled ligand. The choice of brand depends on the sensitivity required, the ease of conjugation with the compound, the stability requirements and the available instrumentation. For a review of the different brands or signal producing systems that may be used, see U.S. Patent No. 4,391,904, which is incorporated herein by reference. Antibodies reactive with a specific protein can also be measured through a variety of immunoassay methods. For a review of the immunoassay and immunoassay procedures applicable to measurement-the antibodies by immunoassay techniques, see Stites and Terr (eds.) Basic and Clinical Immunology (7th ed.) Supra.; Maggio (ed.) Enzyme Immunoassay, supra; and Harlow and Lane Antibodies, A Laboratory Manual, supra. In short, the immunoassays for measuring antisera reactive with CX3Cucine proteins can be competitive or non-competitive binding assays. In competitive binding assays the analyte sample competes with an analyte labeled by the specific binding sites in a capture agent bound to a solid surface. Preferably, the capture agent is a purified, recombinant CX3 protein produced as described above. It is also possible to use other sources of CX3 proteins, including protein that occurs in nature, isolated or partially purified. Non-competitive assays include sandwich assays, in which the analyte sample binds between two analyte-specific binding reagents. One of the binding agents is used as a capture agent and attached to a solid surface. The second labeling agent is used to measure or detect the resulting complex by visual means or by instrument. It is possible to use various combinations of the capture agent and labeled binding agent. A variety of different immunoassay formats, separation techniques and labels can also be used similar to those described above for the measurement of CX3Cucine proteins.

SAW. CX3 Purified chambers The amino acid sequences of CX3 Human are given in SEQ ID NOs 2 and 4. The nucleotide and mouse amino acid sequences are given in SEQ ID NOs 5, 6, 7 and 8. The purified protein or peptides defined they are useful for generating antibodies by normal methods, as described above. Synthetic peptides or purified protein, for example, the chemokine domains may be present for an immune system to generate polyclonal and monoclonal antibodies. See, for example, Coligan (1991) Current Protocols in Immunology Wiley / Greene, NY; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY, which are incorporated herein by reference. Otherwise, a CX3Cycin receptor may be useful as a specific binding reagent and one may take advantage of this binding specificity, for example, purification of a CX3Cycin ligand. The specific binding composition can be used to screen an expression library made from a cell line expressing a CX3Cycin. Various methods are available for screening, for example, standard staining of the ligand expressed on the surface or by panning technique. Screening of intracellular expression can also be performed by the various staining or immunofluorescence procedures. The binding compositions can be used to affinity purify or classify the cells expressing the ligand. The segments of the peptide, together with the comparison with the homologous genes, can also be used to produce the oligonucleotides suitable for screening a library. The genetic code can be used to select suitable oligonucleotides useful as probes for screening. In combination with polymerase chain reaction (PCR) techniques, synthetic oligonucleotides will be useful for selecting the desired clones from a library, including natural allelic and polymorphic variants.

Peptide sequences allow the preparation of peptides to generate antibodies to know these segments, and allow the preparation of oligonucleotides encoding these sequences. The sequence also allows for synthetic preparation, for example, see Dawson, et al. (1994) Science 266: 776-779. Since the CX3Cucines appear to be soluble proteins, the gene will usually possess an N-terminal signal sequence, which is eliminated by processing and secretion, and the putative cleavage site is between amino acids 24 (GLY) and 25 (GLN) ) in SEC ID NO: 2 or 4, although it can be slightly in any direction. The analysis of structural features compared to the sequences reported as more closely related has shown similarities with other cytokines, particularly the class of proteins known as chemokines. Within the chemokines are two subgroups, the CC and CXC subgroups. The CX3Ccinas family shares some characteristics with each of these groups, but its combination of characteristics is distinctive and defines a new family of related chemokines. While other structural features are the result of the sequences provided in SEQ ID NOs 1 through 8, the characteristic "chemokine in a stem" is provided by the stem region possessing various sites that can provide a highly glycosylated domain. Stem structure may be important in the presentation of CX3C chemokine to other cells. In fact, it seems that the stem region can be processed to release the soluble chemokine. This suggests the possibility of substituting the CX3C chemokine domain with other chemokines to effect efficient presentation against suitable target cells. In addition, the "stem" regions are likely to affect the solubility and pharmacological aspects of the protein. As such, this region will be the target of analysis to evaluate and modulate these characteristics as pharmacokinetics. Truncation of this protein can affect the half-life, elimination and accessibility of the chemokine domains.

VII. PHYSICAL VARIANTS This invention also encompasses proteins or peptides that have substantial similarity in the sequence of amino acids with an amino acid sequence of a CX3Cinema Natural variants include individual, polymorphic, allelic, strain or species variants. The similarity in the sequence of amino acids or the identity of the sequence is determined by optimizing the couplings of the residues, if necessary, introducing spaces as required. This changes when conservative substitutions are considered as couplings. Conservative substitutions usually include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine, lysine, arginine; and phenylalanine, tyrosine. Homologous amino acid sequences include natural, polymorphic, allelic and interspecies variants in each respective protein sequence. Common homologous proteins or peptides will have from 50-100% similarity (if spaces can be introduced), up to 75-100% similarity (if conservative substitutions are included) with the amino acid sequence of CX3Cucines. The measures of similarity will be at least about 50%, in general at least 60%, more generally at least 65%, by regular at least 70%, more regularly at least 75%, preferably at least 80% % and more preferably at least 80%, and in the particularly preferred embodiments, at least 85% or more. See also Needleham, et al. (1970) J. Mol. Biol. 48: 443-453; Sankoff, et al. (1983) Time Warps, String Edits, and Macromolecules: The Theory and Practice of Sequence Comparison, Chapter 1, Addison-Wesley, Reading, MA; and software packages from IntelliGenetics, Mountain View, CA; and the University of Wisconsin Genetics Computer Group, Madison, Wl. Nucleic acids encoding CX3 mammalian protein will usually hybridize to the nucleic acid sequence of SEQ ID NO: 1, 3, 5 or 7 under severe conditions. For example, nucleic acids encoding human CX3 proteins will usually hybridize to the nucleic acid of SEQ ID NO 1 under severe hybridization conditions. In general, severe conditions are selected around 10 ° C less than the thermal melting point (Ft) for the probe sequence at a defined ionic strength and pH. The Ft is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly coupled probe. Typically, severe conditions will be those in which the salt concentration is about 0.2 molar at pH 7, and the temperature is at least about 50 ° C. Other fractions can significantly affect the severity of hybridization, including, among others, the base composition and the size of the complementary strands, the presence of organic solvents such as formamide, and the magnitude of the base decoupling. A preferred embodiment will include nucleic acids that will bind to the sequences described in 50% formamide and 200 mM NaCl at 42 ° C.

An isolated CX3Ccina DNA can be easily modified by nucleotide substitutions, nucleotide depletions, nucleotide insertions or short insertions of nucleotide elongations. These modifications will result in novel DNA sequences encoding antigens for CX3Cacinas, their derivatives or proteins having highly similar antigenic or immunogenic physiological activity. It is possible to use modified sequences to produce mutant antigens or to improve expression. Improved expression can include genetic amplification, increased transcription, increased translation, and other mechanisms. These mutant derivatives of CX3Ccina include predetermined or site-specific mutations of the respective protein or its fragments. The "mutant CX3Ccina" comprises a polypeptide that would otherwise fall within the homology definition of the human CX3Cina as set forth above, but it has an amino acid sequence that differs from a CX3Cine as it is found in nature, either through depletion, substitution or insertion. In particular, the site-specific mutant CX3Cina "generally includes proteins that have significant similarity to a protein having a sequence of SEQ ID NO: 2, 4, 6 or 8 and shares various biological activities, eg, antigenic or immunogenic , with these sequences, and in the preferred modalities they contain most or all of the sequence described.This also applies to polymorphic variants from different individuals.Similar concepts apply to different CX3Cucine proteins, specifically those found in various animals. of warm blood, for example, mammals and birds.As already stated, it is emphasized that the descriptions generally comprise other CX3Cine proteins, not limited to the human or mouse modalities specifically described. sites are predetermined, the mutants do not need to be site-specific. Sis of the CX3Ccina can be carried out by making insertions or depletions of amino acids. Substitutions, depletions, insertions or any combination can be generated, to arrive at a final construction. These include substitution levels of amino acid residues from none, one, two, three, five, seven, ten, twelve, fifteen, etc. The insertions include terminal amino or carboxyl fusions, for example, epitope tags. Random mutagenesis can be carried out in an objective codon and the expressed mutants can then be screened for the desired activity. Methods for carrying out substitution mutations at predetermined sites in DNA having a known sequence are well known in the art, for example, by mutagenesis of the M13 primer or polymerase chain reaction (PCR) techniques. See also, Sambrook, et al. (1989) and Ausubel, et al. (1987 and supplements). Mutations in DNA normally should not be carried out in coding sequences between reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structure such as loops or hairpins. The present invention also provides reccmbinant proteins, for example, heterologous fusion proteins using segments derived from these proteins, both CX3Cine or antigen-binding sites. A heterologous fusion protein is a fusion of proteins or segments that normally do not fuse in nature in the same way. In this manner, the product of the fusion of an immunoglobulin with a CX3Cycin polypeptide is a continuous protein molecule having sequences fused to a common peptide bond, usually made as an individual translation product and having properties derived from each one of the origin peptides. A similar concept applies to heterologous nucleic acid sequences.

In addition, it is possible to elaborate new constructions combining similar functional domains of other proteins. For example, protein-binding segments or other segments can be "exchanged" between new polypeptides or different fusion fragments. See, for example, Cunningha, et al. (1989) Science 243: 1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263: 15985-15992. In this way, novel chimeric polypeptides that present new combinations of specificities will result from the functional binding of protein-binding specificities and other functional domains.

VIII. LINKING AGENT: COMPLEXES OF CX3 PROTEIN A CX3 protein that specifically binds or that specifically immunoreacts with an antibody raised against a defined immunogen, such as an immunogen containing the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, is usually determined in an immunoassay. The immunoassay uses a polyclonal antiserum that was developed for a protein of SEQ ID NO. 2, 4, 6 or 8. This antiserum is selected with little cross-reactivity against other chemokines and any of this cross-reactivity is eliminated by immunoabsorption before being used in the immunoassay.

To produce antisera for use in an immunoassay, the protein of SEQ ID NO: 2, 4, 6 or 8 is isolated as described herein. For example, the recombinant protein can be produced in a mammalian cell line. A mouse strain reproduced such as balb / c is immunized with the protein of SEQ ID NO: 2, 4, 6 or 8, using standard adjuvants, such as Freund's adjuvant and a standard mouse immunization protocol (see Harlow and Lane, supra). In an alternative mode, a synthetic peptide, preferably of almost complete length, from the sequences described in the present invention and conjugated to a carrier protein can be used an immunogenic [sic]. The polyclonal sera are collected and titrated against the immunogenic protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titre of 104 or greater are selected and tested for cross-reactivity against C, CC and CX3C chemokines using a competitive binding immunoassay, such as the one described in Harlow and Lane, supra, on pages 570 -573. Preferably two chemokines are used in this determination together with CX3 human CX3 or CX3 mouse.

Together with a CX3Ccina, the monocyte chemotactic protein-1-MCP-1) and the inflammatory-macrophage protein (Mip-la) are used to identify antibodies that are specifically bound to a CX3Cine. Together with the human CX3Cucines, monocyte chemotactic protein-2 (MCP-2) and Mip-la are used to identify antibodies that are specifically bound to a CX3Cine. These chemokines can be produced as recombinant proteins and can be isolated using standard molecular chemistry and protein chemistry techniques as described herein. Immunoassays in the unior format. Competitiveness can be used for cross-ration determinations. For example, a protein of SEQ ID NO: 2, 4, 6 or 8 can be immobilized on a solid support. The proteins added to the test compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to the protein of SEQ ID NO: 2.4, 6 or 8. The percentage of cross-reactivity for the above-mentioned proteins is calculated using the normal calculations Antisera with less than 10% cross-reactivity with each of the proteins mentioned above are selected and combined. The cross-reactive antibodies are then removed from the combined antisera by immunoabsorption with the aforementioned proteins. The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein to the immunogenic protein (e.g., the chemokine motif of CX3Cine of SEQ ID NO: 2. 4, 6 or 8). To make this comparison, the two proteins are each tested in a wide range of concentrations and the amount of each protein necessary to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein needed is less than twice the amount of the protein of SEQ ID NO: 2 that is required, then the second protein is said to bind specifically to an antibody generated for the immunogen. It should be understood that CX3Ccina proteins are a family of homologous proteins that comprise two or more genes. For a specific gene product, such as the human CX3 protein, the term refers not only to the amino acid sequences described herein, but also to other proteins that are polymorphic, allelic, non-allelic, or species variants. It should also be understood that the term "CX3 human CX3 or CX3 mouse" includes unnatural mutations introduced by deliberate mutations using conventional recombinant technology such as mutation at a single site, or by cutting small sections of DNA encoding CX3Cacinas proteins or substituting new amino acids or adding new amino acids. These minor alterations must substantially maintain the immunoidentity of the original molecule and / or its biological activity. In this manner, these alterations include proteins that are specifically immunoreactive with a CX3 protein such as occurs in nature, for example, the CX3 protein human protein as shown in SEQ ID NO: 2 or 4. The biological properties of the altered proteins they can be determined by expressing the protein in a suitable cell line and measuring, for example, the chemotactic effect. Specific modifications of the protein considered minor will include the conservative substitution of amino acids with similar chemical properties, as described above for the CX3Ccína family as a whole. By optimally aligning a protein with the protein of SEQ ID NO: 2. 4, 6 or 8 and using conventional immunoassays as described herein to determine immunoidentity, or using lymphocyte chemotaxis assays, it is possible to determine the protein compositions of the invention.

IX. Functional Variants Blocking the physiological response to CX3Cycins may result from inhibiting the binding of the protein to its receptor, for example, by competitive inhibition. Thus, in vitro assays of the present invention will often use isolated protein, membranes from cells expressing a recombinant membrane associated with CX3Cine, soluble fragments containing receptor binding segments of these proteins or fragments bound to in-phase substrates. solid These assays will also allow the diagnostic determination of the effects of mutations and modifications of the binding segments or mutations and modifications of the protein, for example protein analogues. This invention also contemplates the use of competitive drug screening assays, for example, wherein the neutralizing antibodies to the antigen or receptor fragments compete with a test compound to bind to the protein. In this form, the antibodies can be used to detect the presence of a polypeptide that shares one or more antigen-binding sites, of the protein, and can also be used to occupy binding sites on the protein that can otherwise interact. with a receiver. "Derivatives" of the antigens of CX3Ccina includes mutants of the amino acid sequence, glycosylation variants and covalent conjugates or aggregates with other chemical portions. Covalent derivatives can be prepared by linking the functionalities to the groups found on the side chains of the amino acids of CX3Cine or N- or C-terminal, by means which are well known in the art. These derivatives may include, without limitation, aliphatic esters or carboxyl terminal amides or residues containing carboxyl side chains, o-acyl derivatives of hydroxyl group-containing residues and N-acyl derivatives of the amino-terminal amino acid-containing residues or amino groups, for example lysine or alginin. The acyl groups are selected from the group of alkyl portions including normal alkyl of C3 to C18, thereby forming alkanoylaryl species, see, for example, Coligan, et al. (eds.) (1995 and periodic supplements) Current Protocols in Protein Science, John Wiley and Sons, New York, NY. Covalent binding to carrier proteins can be important when the immunogenic portions are haptens. In particular, alterations to glycosylation are included, for example, carried out by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in other processing steps. Particularly preferred means to accomplish this is by exposing the polypeptide to glycosylating enzymes from cells that normally provide this processing, for example, mammalian glycosylation enzymes. Enzymes of deglucosilation are also contemplated. Also included are versions of the same primary amino acid sequence having other minor modifications, including phosphorylated amino acid residues, for example phosphotyrosine, phosphoserine or phosphothreonine or other portions, including ribosyl groups or cross-linking reagents. An important group of derivatives are covalent conjugates of CX3Cine or fragments thereof with other proteins or polypeptides. These derivatives can be synthesized in recombinant culture such as N- or C-terminal fusions or by the use of agents known in the art for their utility in cross-linking proteins by reactive side groups. Preferred protein derivatization sites with crosslinking agents are found in free amino groups, carbohydrate moieties and cysteine residues. Fusion polypeptides between CX3Kines and other homologous or heterologous proteins are also provided. Various growth factors and cytokines are homodimeric entities and a repeat construct can have various advantages, including decreased susceptibility to proteolytic degradation. In addition, many receptors require dimerization to transduce a signal, and various dimeric proteins or domain repeats may be desirable. The heterologous polypeptides can be fusions between different surface markers, resulting in, for example, a hybrid protein exhibiting receptor binding specificity. Likewise, it is possible to construct heterologous fusions that present a combination of properties or activities of the derived proteins. Common examples are fusions of a reporter polypeptide, e.g., luciferase, with a segment or domain of a protein, e.g., a receptor binding segment, so that the presence or location of the fused protein can be easily determined . See, for example, Dull, et al., U.S. Patent No. 4,859,609. Other gene fusion patterns include bacterial β-galactosidase, trpE, Protein A, β-lactamase, alpha amylase, alcohol dehydrogenase and yeast coupling factor. See, for example, Dawson, et al. (1994) Science 266: 776-779; and Godowsky, et al. (1988) Science 241: 812-816. In particular, fusion proteins with portions from the related genes will be useful. Similar concepts of fusions with antigen-binding sites are contemplated. These polypeptides may also have amino acid residues that have been chemically modified by phosphorylation, sulfonation, biotinylation or the addition or removal of other portions, particularly those having molecular forms similar to phosphate groups. In some embodiments, the modifications will be useful in labeling reagents or serve as purification targets, for example affinity ligands. This invention also contemplates the use of derivatives of CX3Cinas different from variations in amino acid sequence or glycosylation. These derivatives may include covalent or aggregative association with chemical portions. These derivatives include: (1) salts, (2) covalent modifications of terminal residues and side chains, and (3) adsorption complexes, for example with cell membranes. These covalent or aggregative derivatives are useful as immunogens, as reagents in immunoassays or in purification methods such as for the affinity purification of ligands or other binding ligands. For example, a CX3Cycin antigen can be immobilized by covalent attachment to a solid support such as SEPHAROSE activated with cyanogen bromide, by methods that are well known in the art, or adsorbed onto polyolefin surfaces, with or without crosslinker glutaraldehyde, for use in the assay or purification of anti-CX3 antibodies or its receptor. The CX3Ccines can also be labeled with a detectable group, for example, they can be labeled with iodine radiated by the chloramine T procedure, they can be covalently bound to rare earth chelates or they can be conjugated to another fluorescent portion for use in assays of diagnosis. The purification of the CX3Ccinas can be carried out by antibodies or immobilized receptor. The isolated CX3Ccina genes will allow the transformation of cells lacking pressure from the corresponding CX3Cucines, for example, types or cells of species lacking corresponding proteins and exhibiting negative background activity. The expression of transformed genes will allow the isolation of antigenically pure cell lines, with variants of defined or unique species. This approach will allow more sensitive detection and discrimination of the physiological effects of CX3Cine receptor proteins. The subcellular fragments, for example cytoplasts or fragments of membranes, can be isolated and used.

X. USES The present invention provides reagents that will find use in diagnostic applications as described elsewhere herein, for example, in the general description for developmental abnormalities or below in the description of diagnostic kits.

The nucleotides of CX3Ccina, for example, the DNA or RNA of CX3 Human or mouse can be used as a component in a forensic assay. For example, the provided nucleotide sequences can be labeled using, for example, 32P or biotin and can be used to test strips of polymorphism of standard restriction fragments, providing a measurable character to help distinguish between individuals. These probes can be used in well-known forensic techniques such as genetic fingerprinting. In addition, nucleotide probes prepared from CX3Cycin sequences can be used in in situ assays to detect chromosomal abnormalities. For example, rearrangements in the mouse chromosome encoding a CX3Ccina gene can be detected through well-known in situ techniques, using CX3Ccina probes along with other well-known chromosomal markers. Antibodies and other binding agents directed towards CX3 proteins or nucleic acids can be used to purify the corresponding CX3 molecule. As described in the following examples, the purification of antibodies from the CX3Cine components is possible and practicable. The antibodies and other binding agents can also be used in a diagnostic mode to determine if the CX3Cine components are present in a tissue sample or cell population using well-known techniques as described herein. The ability to bind a binding agent to a CX3Cine provides a means to diagnose disorders associated with poor CX3Cine regulation. Antibodies and other CX3Cine binding agents may also be useful as histological markers. As described in the following examples, the expression of CX3Cine is limited to specific tissue types. By directing a probe, such as an antibody or nucleic acid to a CX3Cine, it is possible to use the probe to distinguish tissue types and cells in situ or in vitro. This invention also provides reagents with significant therapeutic value. CX3Cine (in their natural or recombinant state), fragments of these and antibodies for these, together with compounds identified as having binding affinity to a CX3Cine, are useful in the treatment of conditions associated with physiology or abnormal development, including Abnormal proliferation, for example, cancerous states or degenerative states. Abnormal proliferation, regeneration, degeneration and atrophy can be modulated by suitable therapeutic treatment using the compositions provided herein. For example, a disease or disorder associated with abnormal expression or abnormal signaling by a CX3Cine is a target for a protein agonist or antagonist. In the same way, proteins play a role in the regulation or development of neuronal or hematopoietic cells, for example, lymphoid cells, which affect immune responses. Other abnormal developmental states are known in cell types that show to possess CX3Ccina RNA by Northern blot analysis. See, Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway, NJ; and Thorn, et al. Harrison s Principies of Interna! Medicine, McGraw-Hill, NY. Developmental or functional abnormalities, for example of the neuronal or immune system, cause significant medical abnormalities and conditions that may be susceptible to prevention or treatment using compositions as provided herein. Certain chemokines have also been implicated in viral replication mechanisms. See, for example Cohen (1996) Science 272: 809-810; Feng, et al. (1996) Science 272: 872-877; and Cocchi, et al. (1995) Science 270: 1811-1816. The CX3C chemokine may be useful in a similar context. Alternatively, the stem structure can be very important in the presentation of the ligand domain, and other chemokines can be advantageously substituted by the chemokine domain in this molecule. Modification in the "stem" structure can affect many of the pharmacological properties of CX3Cine, including half-life and biological activity. The recombinant CX3 or CX3Cine antibodies can be purified and then administered to a patient, for example, in sterile form. These reagents can be combined for therapeutic use with additional active or inert ingredients, for example, in conventional pharmaceutically acceptable carriers or diluents, for example, immunogenic adjuvants, together with physiologically harmless stabilizers and excipients. These combinations can be filtered in sterile form and it is possible to put them in dosage form as by lyophilization in small bottles for the dose or they can be stored in stabilized aqueous preparations. This invention also contemplates the use of antibodies or binding fragments thereof, including forms that do not bind to complement. Screening of the drug using antibodies or the receptor or fragments thereof may identify compounds that have binding affinity to the CX3Cenzas, including the isolation of associated components. Subsequent biological assays can then be used to determine if the compound has intrinsic stimulating activity and is therefore a blocker or antagonist since it blocks the activity of the protein. In the same way, a compound that has intrinsic stimulating activity can activate the receptor and, in this way, is an agonist because it stimulates the activity of a CX3Cine. This invention also contemplates the therapeutic use of antibodies to CX3Ccina as antagonists. This approach should be particularly useful with other variants of CX3Cine species. The amounts of reagents needed for effective therapy will depend on multiple different factors, including the means of administration, target site, physiological status of the patient and other medications administered. In this way, the doses of the treatment must be titrated to optimize safety and efficacy. In general, doses used in vitro can provide useful guidance in amounts useful for the administration of these reagents in situ. Tests of effective doses in animals for the treatment of specific disorders will provide other predictive indications of the dose in humans. Various considerations are described in, for example, Gilman, et al. (eds.) (1990) Goodman and Gilman s: The Pharmacological Bases of Therapeutics (8th ed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences (17th ed.) Mack Publishing Co., Easton, PA. Methods for administration are described now and in the following, for example for oral, intravenous, intraperitoneal or intramuscular administration, transdermal diffusion and others. The pharmaceutically acceptable carriers will include water, saline, buffers and other compounds described, for example, in Merck Index, Merck & Co., Rahway, NJ. Dosage ranges will usually be expected in amounts below concentrations of 1 mM, usually lower concentrations of about 10 μM, usually less than about 100 nM, preferably less than about 10 pM (picomolar), and more preferably less than about IfM (femtomolar), with a suitable carrier. Slow release formulations, or an apparatus for slow release will often be used for continuous administration. The CX3Ccinas, fragments of this and antibodies for this or its fragments, antagonists and agonistscan be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to the carrier proteins such as ovalbumin or serum albumin before administration. Therapeutic formulations can be administered in multiple conventional dosage formulations. Although it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. The formulations will usually comprise at least one active ingredient, as stated above, together with one or more acceptable carriers thereof. Each carrier must be pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not harming the patient. The formulations include those suitable for oral, rectal, nasal or parenteral administration (including subcutaneous, intramuscular, intravenous and intradermal). The formulations can be presented, conveniently, in unit dosage forms and can be pre-prepared by any of the methods well known in the pharmacy art. See, for example, Gilman, et al. (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics (8th ed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences (17th ed.) Mack Publishing Co., Easton, PA; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, NY. The therapy of this invention can be combined with or used in association with other therapeutic agents. The forms in their natural state and the recombinants of the CX3Ccines of this invention are particularly useful in kits and assay methods that are capable of screening compounds for protein binding activity. In recent years, various automated testing methods have been developed to allow the screening of tens of hundreds of compounds in a short period. See, for example Fodor, et al. (1991) Science 251: 767-773, and other descriptions of chemical diversity libraries, which describe means for testing binding affinity by a plurality of compounds. The development of suitable assays can be greatly facilitated by the availability of large amounts of purified, soluble CX3Cine as that provided by this invention. For example, antagonists can usually be found once the protein has been structurally defined. Now it is possible to test the potential analogs of the protein with the development of highly automated assay method using a purified receptor. In particular, new agonists and antagonists will be discovered using screening techniques as described herein. Of specific importance are the compounds found with a combined binding affinity for multiple CX3Cine receptors, for example compounds that can serve as antagonists for species variants of a CX3Cine. This invention is particularly useful for screening compounds using recombinant protein in a variety of drug screening techniques. The advantages of using a recombinant protein in screening for specific ligands include: (a) improved renewable source of CX3Cinema coming from a specific source; (b) a potentially larger number of ligands per cell giving a better signal-to-noise ratio in the assays; and (c) specificity of the species variant (theoretically giving greater biological and disease specificity). A drug screening method uses eukaryotic or prokaryotic host cells that are stably transformed with recombinant DNA molecules that express a CX3Cycin receptor. It is possible to isolate cells that express a receptor during the isolation of any other. These cells, in variable or fixed form, can be used for normal ligand / receptor binding assays. See also, Parce, et al. (1989) Science 246: 243-247; and Owicki, et al. (1990) Proc. Nat'l Acad. Sci. USA 87: 4007-4011, which describes sensitive methods for detecting cellular responses. Competitive assays are particularly useful, where the cells (source of CX3Cine) are contacted and incubated with a labeled receptor or antibody having known binding affinity to the ligand, such as 125i-antibody, and a test sample. whose affinity of union to the composition is going to be measured. The bound and free labeled binding compositions are then separated to assess the degree of ligand binding. The amount of binding of the test compound is inversely proportional to the amount of binding to the labeled receptor at the known source. It is possible to use any of the numerous techniques to separate bound ligands from free ligands to assess the degree of ligand binding. This separation step usually includes a procedure such as adhesion to the filters followed by washing, adhesion to plastics followed by washing or centrifugation of the cell membranes. Viable cells can also be used to screen for the effects of drugs on the functions mediated by CX3Ccina, for example, levels of the second messenger, ie, Ca ++; cell proliferation; changes in the inositol phosphate bank and inositol phosphate and others. Some detection methods allow the elimination of a separate step, for example, a detection system sensitive to proximity. Calcium-sensitive dyes will be useful for detecting Ca ++ levels, with a fluorimeter or a cell sorting device by fluorescence. Another method uses membranes from transformed eukaryotic or prokaryotic host cells as the source of a CX3Cine. These cells are stably transformed with DNA vectors directing the expression of a CX3Cycina, for example, a designed membrane-bound form. In essence, the membranes would be prepared from the cells and used in a receptor / ligand binding assay such as the competitive assay established in the above. Still another method is to use CX3 solubilized, unpurified or solubilized, purified Cucina from transformed eukaryotic or prokaryotic host cells. This allows a "molecular" binding assay with the advantages of increasing the specificity, the ability to automate and high performance of the drug test. Another technique for screening drugs includes a method that provides high throughput screening for compounds that have adequate binding affinity for a CX3Cycin antibody and is described in detail in Geysen, European Patent Application 84/03564, published September 13, 1984 First, large amounts of small, different, peptide test compounds are synthesized on a solid substrate, for example, plastic pins or some other suitable surface, see Fodor, et al., Supra. Then all the pins are reacted with solubilized, purified, solubilized, purified, CX3Ccin antibodies, and washed. The next step includes detection of the antibody binding to CX3Cine.

The rational design of the drug may also be based on structural studies of the molecular forms of CX3Cine and other effectors or analogues. See, for example, Methods in Enzymology vols. 202 and 203. The effectors may be other proteins that mediate other functions in response to binding to the ligand or other proteins that normally interact with the receptor. A means to determine the sites that interact with other specific proteins is a physical determination of the structure, for example X-ray crystallography or two-dimensional NMR techniques. These will offer a guide to know which amino acid residues form molecular contact regions. For a detailed description of the structural determination of the protein, see, for example Blundell and Johnson (1976) Protein Crystallography Academic Press, NY. A purified CX3Cin can be coated directly onto plates for use in the aforementioned drug screening techniques. However, it is possible to use non-neutralizing antibodies for these ligands as capture antibodies to immobilize the respective ligand on the solid phase.

XI: KITS This invention also contemplates the use of CX3Ccina proteins, fragments thereof, peptides and their fusion products in a variety of diagnostic kits and methods for detecting the presence of CX3Ccina or a CX3Ccina receptor. Typically, the kit will have a compartment containing a defined CX3 peptide or gene segment or a reagent that recognizes one or the other, eg, receptor fragments or antibodies. A kit for determining the binding affinity of a test compound for a CX3Cine will usually contain a test compound; a labeled compound, for example, a receptor or antibody having known binding affinity for CX3Cine; a source of CX3Cine (in its natural or recombinant state); and a means for separating the free labeled compound from the bound, such as a solid phase, to immobilize the CX3Cine. Once the compounds are screened, those that have adequate binding affinity to CX3Ccina can be evaluated in suitable biological assays, as known in the art, to determine whether they act as agonists or antagonists to the receptor. The availability of the recombinant CX3Cine polypeptides also offer well-defined standards for calibrating these assays.

A preferred kit for determining the concentration of, for example, a CX3Cine in a sample will usually contain a labeled compound, eg, receptor or antibody, which has known binding affinity for CX3Cine, a source of CX3Cine (in nature) or recombinant), and a means for removing the labeled compound bound from the free, for example a solid phase to immobilize the CX3Cine. The compartments containing the reagents, and instructions will normally be provided. Antibodies, including antigen-binding fragments, specific for CX3Cine or ligand fragments are useful in diagnostic applications to determine the presence of elevated levels of CX3Cine and / or its fragments. This can allow diagnostics of the quantities of the differently processed forms of CX3Cine, for example, stems structure successively degraded, these diagnostic assays can employ Used, living cells, fixed cells, immunofluorescence, cell cultures, body fluids and can also influence the detection of antigens related to the ligand in serum, or the like. The diagnostic tests can be homogeneous (without a separation step between free reagent and the antigen-CX3Cine complex) or heterogeneous (with a separation step). There are several commercial assays, such as the radioimmunoassay (RIA), the enzyme-linked immunosorbent assay (ELISA), the enzyme immunoassay (EIA), the enzyme-linked immunoassay (EMIT), the fluorescent immunoassay of the labeled substrate (SLFIA). and similar. For example, unlabeled antibodies can be using a second antibody that is labeled and that recognizes the antibody for a CX3Cine or a specific fragment thereof. Similar trials have also been widely described in the literature. See, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press, NY; Chan (ed.) (1987) Immunoassay: A Practical Guide Academic Press, Orlando, FL; Price and Newman (eds.) (1991) Principles and Practice of Immunoassay Stockton Press, NY; and Ngo (ed.) (1988) Nonisotopic Immunoassay Plenum Press, NY. Anti-idiotypic antibodies may have similar use to diagnose the presence of antibodies against a CX3Cine, as such it may be diagnostic of different abnormal states. For example, the overproduction of CX3Ccina may result in the production of various immunological reactions or other medical reactions that may be diagnostic of abnormal physiological states, for example, in the growth, activation or differentiation of the cells.

Reagents for diagnostic tests are often supplied in kits or equipment to optimize the sensitivity of the assay. For the present invention, depending on the nature of the assay, the protocol and the label, the labeled or unlabeled antibody or receptor or labeled CX3Cina is provided. This is usually together with other additives, such as buffer solutions, stabilizers, materials necessary for the production of the signal, such as substrates for enzymes, and the like. Preferably, it will also contain instructions for proper use and to dispose of the contents after use. Usually, the kit has compartments for each useful reagent. It is desirable that the reagents be provided as a dry lyophilized powder, wherein the reagents can be reconstituted in an aqueous medium that provides the appropriate concentrations of the reagents to perform the assay. Most of the aforementioned constituents of drug screening and diagnostic assays can be used without modification, or can be modified in a variety of ways. For example, labeling can be carried out by covalent or non-covalent attachment of a portion that directly or indirectly provides a detectable signal. In any of these assays, the protein, the test compound, the CX3Cine or antibodies thereto can be directly or indirectly labeled. The possibilities for direct labeling include label groups: radiolabelled with I5I, enzymes, (US Patent No. 3,645,090) such as alkaline phosphatase and peroxidase and fluorescent labels (US Patent No. 3,940,475) capable of monitoring the change in fluorescence intensity , displacement of the wavelength or polarization of the fluorescence. The possibilities for indirect labeling include biotinylation of a constituent, followed by avidin binding coupled to one of the above labeled groups. There are also various methods for separating the bound ligand from the free or, otherwise, the bound test compound from the free. The CX3Ccina can be immobilized on various matrices followed by washing. Suitable matrices include plastics such as an ELISA plate, filters and beads, methods for immobilizing CX3Cine in a matrix include, without limitation, direct adhesion to the plastic, the use of capture antibody, chemical coupling and biotin-avidin. The last step in this approach includes the precipitation of the ligand / receptor or ligand / antibody complex by any of the various methods including those used, an organic solvent such as polyethylene glycol or a salt such as ammonium sulfate. Other suitable separation techniques include, without limitation, the magnetizable antibody fluorescence particle method, as described in Ra et al. (1984) Clin. Chem. 30: 1457-1461. And separation of the magnetic particle from the double antibody, as described in U.S. Patent No. 4,659,678. Methods for linking proteins or their fragments to the various brands has been widely reported in the literature and does not require a more detailed description in the present. Most of the techniques include the use of carboxyl groups activated by the use of carbodiimide or active esters to form the peptide bonds, the formation of thioethers by the reaction of a mercapto group with activated halogen such as chloroacetyl, or an activated olefin as maleimide, for the link, or similar. The fusion proteins will also find use in these applications. Another aspect of the diagnosis of this invention includes the use of oligonucleotide or polynucleotide sequences formed from the sequence of a CX3Cycin. These sequences can be used as probes to detect levels of CX3Ccina messages, in samples from natural sources, or of patients suspected of having an abnormal state, for example, cancer or a developmental problem. The preparation of the RNA and DNA nucleotide sequences, the labeling of the sequences and the preferred site of the sequences has received extensive description and discussion in the literature. Ordinarily, an oligonucleotide probe must have at least about 14 nucleotides, usually at least about 18 nucleotides, and the polynucleotide probes can be up to several kilobases. Various brands can be used, the most common being radionuclides, particularly 3iP. However, it is also possible to employ other techniques, such as the use of biotin-modified nucleotides for introduction into a polynucleotide. Biotin then serves as the site for binding to avidin or antibodies, which can be labeled with a wide variety of labels, such as radionuclides, fluorophores, enzymes or the like. Otherwise, it is possible to use antibodies that can recognize specific duplexes, including DNA duplexes, RNA duplexes, hybrid DNA-RNA duplexes, or DNA-protein duplexes. The antibodies in turn can be labeled and the assay can be carried out where the duplex is bound to a surface so that, with the formation of the duplex on the surface, the presence of antibody binding to the duplex can be detected. . The use of probes for novel anti-sense RNA can be carried out using various conventional techniques, such as nucleic acid hybridization, plus and minus screening, recombinatory probing, hybrid liberated translation (HRT), and translation captured at hybrid (HART). This also includes the amplification techniques such as the polymerase chain reaction (PCR). Diagnostic kits that also test the qualitative or quantitative presence of other markers is also contemplated. The diagnosis or prognosis may depend on the combination of multiple indications used as markers. In this way, kits can try combinations of markers. See, for example, Viallet, et al. (1989) Progress in Gorwth Res. 1: 89-97.

XII. RECEIVER ISOLATION Having isolated a binding part of a specific interaction, there are methods to isolate the counterpart. See Gearing et al. (1989) EMBO J. 8: 3667-3676. For example, one can determine the means to mark a CX3Cine without interfering with the binding to its receptor. For example, an affinity tag or epitope tag can be fused to the amino- or carboxyl-terminal ligand. An expression library can be screened for specific binding to CX3Cine, for example, by cell sorting or other sieving to detect subpopulations that express this binding component. See, for example, Ho, et al. (1993) Proc Nat '1 Acad. Sic. USA 90: 11267-11271. Otherwise, a panning method can be used. See, for example, Seed and Aruffo (1987) Proc Nat'l Acad. Sic. USA 84: 3365-3369. Also, a two-hybrid selection system can be applied by marking suitable constructions with the available BAS-1 sequences. See, for example, Fields and Song (1989) Nature 340: 245-246. Branded protein crosslinking techniques can be applied to isolate the binding partners or parts of a CX3Cine. This allows the identification of proteins that interact specifically with a CX3Cycin, for example, in a ligand-receptor-like manner. Typically, the chemokine family binds to the receptors of the seven transmembrane receptor family, and the CX3 receptor is likely to have a similar structure. In this way, it is very likely that the receptor is found by expression in a system that is capable of expressing this transmembrane protein in a form capable of presenting ligand binding capacity. The broad scope of this invention will be better understood in connection with the following examples, which have not been proposed to limit the invention to the specific embodiments.

Examples I. General methods Most of the following standard methods are described or have reference, for example, in Maniantis, et al. (1982) Molecular Clongin, A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY; Sambrook et al., (1989) Molecular Clongin, A Laboratory Manual (2nd Ed.) Vols 1-3, CSH_Press, NY; Ausubel, et al., Biology Greene Publishing Associates, Brooklyn NY; o Ausubel, et al. (1987 and supplements) Current Protocols in Molecular Biology Wiley / Greene, NY; Innis, et al. (Eds.) (1990) PCR Protocols: A Guide to Methods and Applications Academic Press, NY. Methods for the purification of proteins include methods such as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization and others. See, Ausubel et al. (1987 and periodic supplements); Deutcher (1990) "Guide to Protein Purification," Methods in Enzymology vol. 182, and other volumes in these series; and the manufacturer's literature on the use of products for protein purification, for example, Pharmacia, Piscataway, NJ, or Bio-Rad, Richmond, CA. The combination with the recombinant techniques allows fusion to the appropriate segments (epitope tags), for example, for a FLAG sequences or an equivalent that can be fused, for example, through a protease-removable sequence. See, for example, Hochuli (1989) Chemische Industrie 12: 69-70; Hochuli (1990) "Purification of Recombinant Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic Engineering, Principie and Methods 12: 87-98, Plenum Press, NY; Crowe, et al. (1992) QIAexpress: The High Level Expression & Protein Purification System QUIAGEN, Inc., Chatswoirth, CA; and Colingan, et al. (Eds.) (1995 and periodic supplements) Current Protocols in Protein Science, John Wiley and Sons, New York, NY. Standard immunological techniques are described, for example, in Coligan (1991) Current Protocols in Immunology, Wiley / Greene, NY; and Methods in Enzymology volumes. 70, 73, 74, 84, 92 93, 108, 116, 121, 132, 150, 162, and 163. Assays for biological activities of neural cells are described in, for example, Wouterlood (ed. 1995) Neuroscience Protocols, Module 10, Elsevier; Methods in Neurosciences Academic Press; and Neuromethods Humana Press,. Totowa, NJ. The methodology of the development systems are described in, for example, Meisa i (ed.) Handbook of Human Growth and Developmental Biology CRC Press; and Chrispeels (ed.) Molecular Techniques and Approaches in Developmental Biology Interscience. FACS analyzes are described in Melamed, et al. (1990) Flow Cytometry and Sorting Wiley-Liss, Inc., New York, NY; Shapiro (1988) Practical Dlow Cytometry Liss, New York, NY; and Robinson, et al. (1993) Handbook of Flow Cystometry Methods Wiley-liss, New York, NY.

II. ISOLATION OF CX3 CLONE A cyclone that encodes human CX3Cine is isolated from a natural source by the very different possible methods. Given the sequences provided herein, primers for PCR or hybridization probes are selected and / or constructed to isolate genomic DNA segments or reverse transcripts of cDNA. The suitable cellular source includes human tissues, e.g., brain libraries. The following tissue distribution also suggests source tissues. Genetic and polymorphic or allelic variants are isolated by screening a population of individuals. RCP-based detection is performed by standard methods, preferably using primers from opposite ends of the coding sequence, but the flank segments can be selected for specific purposes.

Otherwise, probes are selected for hybridization. The specific AT or GC content of the probes is selected depending on the expected homology and the expected decoupling. The appropriate severity conditions are selected to balance an adequate positive background signal ratio. The successive washing steps are used to collect clones of greater homology. Other clones are isolated using a selection procedure based on the antibody. The normal expression cloning methods included, for example, FACS staining of the membrane-associated expression product, are applied. The antibodies are used to identify clones that produce a recognized protein. Otherwise, the antibodies are used to purify a CX3C chemokine, with the sequencing of the protein and the standard means to isolate a gene that encodes this protein. The methods based on the genomic sequence will also allow the identification of naturally available sequences or, otherwise, which have homology for the sequences provided. Similar procedures will result in the isolation of other primate genes.

III. ISOLATION OF CX3C CLAINS FROM RODENT With the above, similar methods are used to isolate a suitable gene for mouse CX3C chemokine. To isolate natural genes, source materials similar to those indicated above are used, including genetic, polymorphic, allelic or strain variants. Variants of species are also isolated using similar methods, for example, rats, moles, muskrats, copybaras [sic], and so on.

IV. ISOLATION OF A CX3 CLONE OF BIRDS As in the previous case, a suitable bird source is selected. Similar methods are used for a variant of species, although the level of similarity will usually be lower for bird CX3C chemokine compared to a human with mouse sequence.

V. EXPRESSION; PURIFICATION; CHARACTERIZATION With a suitable clone, of the aforementioned, the coding sequence is inserted into a suitable expression vector. This may be in a vector specifically selected for an expression system of prokaryote, yeast, insect or higher vertebrate, eg, mammalian. To produce the genetic product standard methods are applied, preferably as a secreted, soluble molecule, but in certain cases it will also be made as an intracellular protein. Intracellular proteins usually require lysis of cells to recover the protein and insoluble inclusion antibodies are a common initial material for further purification. With a clone encoding a vertebrate CX3C chemokine, recombinant production media are used, although the natural forms can be purified from the appropriate sources. Protein product is purified by normal protein purification methods, in certain cases, for example, coupled with immunoaffinity methods. The affinity methods are used as a modification step, as described above, or as a detection assay to determine the separation properties of the protein. Preferably, the protein is secreted into the medium and the soluble product is purified from the medium in a soluble form. Otherwise, as written before, inclusion bodies from prokaryotic expression systems are a source of useful material. Typically, the insoluble protein is solubilized from the inclusion bodies and is replicated using normal methods. The purification methods are developed as described above.

In certain embodiments, the protein is made in a eukaryotic cell which glycosylates the protein in normal form. Purification methods can be affected by this means, such as biological activities. The intact protein can be processed to release the chemokine domain, probably due to a case of protease unfolding somewhere in the stem region, glycosylated, close to the chemokine / stem limit. Although it appears that the recombinant protein is processed, the physiological processes that normally perform this in the native cells must be determined. The product of the purification method described above is characterized to determine multiple structural peculiarities. Various physical methods are applied, for example, amino acid analysis and protein sequences. The resulting protein is subjected to CD spectroscopy and other spectroscopic methods, for example, NMR, ESR, mass spectroscopy, etc. The product is characterized to determine its molecular shape and size, for example, using gel chromatography and the like. The understanding of the chromatographic properties will give rise to more moderate and efficient purification methods. The biochemistry of the CX3C chemokine protein was assessed in mammalian expression systems. Human embryonic kidney 293 cells (HEK 293) transfected with a mammalian expression construct encoding full length CX3C chemokine were etabolically labeled with cysteine and 3oS methionine. The CX3C chemokine was produced as a Mr ~ 95 Kda protein; the transfected control supernatants did not contain these species. Neuraminidase and glucosidases reduced the chemokine Mr CX3C by ~95 Kda ~ 45 Kda, suggesting that the recombinant form is substantially glycosylated. In this manner, the CX3C chemokine cDNA encoding a predicted membrane bound protein encodes a glycoprotein that is released from the cells by an undefined mechanism. The prediction of glycosylation sites can be made, for example, as reported in Hansen et al. (1995) Biochem. J. 308: 801-813.

SAW. PREPARATION OF ANTIBODIES WITH THE CX3CINATION OF VERTEBRATE With the protein product, like the previous one, the animals are immunized to produce antibodies. The polyclonal antiserum is developed using the unpurified antigen, although the resulting serum will have higher background levels. Preferably, the antigen is purified using standard techniques for the purification of proteins, including, for example, affinity chromatography using polyclonal serum as indicated above.

The presence of specific antibodies is detected using defined fragments of synthetic peptides. Preferred fragments include the chemokine domain. The polyclonal serum is developed against a purified antigen, purified as indicated above, or using synthetic peptides. A series of overlapping synthetic peptides comprising the entire full length sequence, without being present in an animal, will produce serum that recognizes most of the linear epitopes on the protein. This antiserum is used for the affinity purified protein, which in turn is used to introduce intact full length protein into another animal to produce another antiserum preparation. Similar techniques are used to generate monoclonal antibodies induced for unpurified antigen or preferably purified antigen.

VII. CELLULAR AND TISSUE DISTRIBUTION The distribution of the protein or the genetic products is determined, for example, using immunohistochemistry with an antibody reagent, as it is produced in the above, or screening nucleic acids that encode chemokine. Hybridization or PCR methods are used to detect DNA, cDNA or message content. Histochemistry allows the determination of specific cell type within a tissue that express higher or lower levels of message or DNA. Antibody techniques are useful for quantifying protein in a biological sample, including a sample of liquid or tissue. Immunoassays are developed to quantify protein. Hybridization techniques were applied to tissue types as seen in Table 2, with positive or negative results as indicated. Commercial strips of tissue may have general contamination of the resident cells, for example, of blood cells or others which populate the tissue. Large or small transcripts correspond to approximate sizes of 4 kb of less than approximately 2 kb, respectively.

Table 2 Distribution in tissue and cell of the human CX3C gene Commercial fabric library: Type of cell Large Small splenic thymus + prostate + + testis + ovary + small intestine + + colon + + Peripheral blood Another analysis of tissue distribution indicates abundance of the human message: heart +++; Brain +++; placenta -; lung ++; liver -; muscle +; Kidney -; pancreas +; spleen -; thymus +; prostate ++; testes +; ovary +; small bowel ++; colon ++; peripherally blood -; line promyelocytic leukemia HL60 -; HeLa S3 cell -; chronic myelogenous leukemia line K562 -; line of lymphoblastic leukemia Molt4 -; RAJ1 line of Burkitts lymphoma; colorectal adenocarcinoma line SW380 +; Lung carcinoma line A549 -; and melanoma line G361 -. "Inverse northerns" are strips of cDNA library with the removed inserts, and size determinations are made based on the size of the inserts in the cDNA library, and manifest the lengths found in the inserts of the cDNA libraries. which may be less than the total length where the reverse transcription was not full length. As such, size determinations do not manifest natural sizes. The results of these are: PBMC (peripheral blood mononuclear cells) +; PBMC (activated using T cell stimulation conditions with anti-CD3 and PMA) -; Mot72 (clone ThO) +; Mot 72 (activated with anti CD28 and anti CD3) and PMA) -; Mot 72a (activated with an antigenic clone, anti peptide) -; Mot 81 (clone ThO latent) -; Mot 81 (activated with anti-CD28 and anti-CD3) -; Hy06 (clone Thl latent) -; Hy06 (activated with anti CD28 and anti CD3) -; Hy06a (activated with angiogenic clone, antipeptide) -; Hy935 (latent Th2 clone) -; Hy935 activated with anti-CD28 and anti-CD3) +; BC bank of transformed EBV + lines; latent splenocytes +; splenocytes + (activated using B cell stimulatory conditions, with anti-CE40 and IL4) -; NK cell bank -; NK bank (activated 6H with PMA and ionomycin) +; NKA6 NK cell clone -; cell clone NKB1 NK -; clone of non-cytotoxic NK + cells; and NK clone stimulated to be cytotoxic -. Other cells and tissues: CHO + cells; Jurkat cells (DNAX) +; Jurkat cells (another source) +; normal T-cell bank 4-; TCT bank (transformed T cells) -; fetal kidney -; fetal lung -; Fetal liver -; fetal heart -; fetal brain +; Fetal gall bladder +; Fetal + small intestine; fetal adipose +; fetal ovary -; fetal uterus +; adult placenta -; fetal + testes; Fetal spleen +; and fetal brain +; additional cells provided: U937 (latent monocyclic cell line) +; C- (monocyte purified by washing or decanted, activated with LPS and IFN- ?, and anti-IL-10) +; C + (activated monocytes decanted with LPS, IFN- ?, and anti-IL-10) +; Ml (activated monocytes decanted with LPS 1H) +; M6 (activated monocytes decanted with LPS 6H) +; 30% DC (30% CDI1 A + dendritic cells, latent, proliferated in TNF-a and GM-CSF) +; 70% DC (70% latent CD1 A + dendritic cells, proliferated in TNF-a and GM-CSF) +; DI (dendritic cells stimulated 1H in PMA and ionomycin) -; D6 (6H stimulated dendritic cells in PMA and ionomycin) -; D5 DC (latent latent dendritic cells cultured 5d in GM-CSF and IL-4) +; DC (dendritic cells cultured in GM-CSF and IL-4, activated with LPS) +; DC (equivalent D5 cells, activated with GM-CSF) +; DC mixture (dendritic cells stimulated with a mixture of cytokines) +; CDla + (CDla + dendritic cells 99% purity enriched from 70% DC) +; CD14 + (fraction CD14 + classified from 70% DC, morphology similar to monocytes) -; CDla + (CDla + and CD86 + at 95% classified from DC to 70%) -; TF1 (hematopoietic precursor line) +; Jurkat (T cell line) +; MRC5 (lung fibroblast sarcoma cell line) +; JT (cell line) +; U937 (premonocyclic cell line) +. Since the endothelium is an important site of chemokine action, a Northern blot test was carried out to assess if the CX3Ccina was expressed in this tissue. The human CX3 was also exposed to be expressed on activated, human primary endothelial cells by expression of mRNA and protein. This suggests that CX3Ccina may be included in the trafficking of leukocytes in various organs. In summary, the CX3 human mRNA was found in monocytes, dendritic cells, T cells and B cells, for example, it was found in certain immune cells.

VIII. MICROCHEMOTAXIS TRIALS The pro-migratory activities of CX3C chemokines have been evaluated in microchemotaxis assays. See, for example, Bacon et al. (1988) Br. J. Pharamacol. 95: 966-974. It appears that CX3C chemokine is a potent attractant of peripheral blood and T cell monocytes. It has been difficult to demonstrate the pro-migratory activity of blood neutrophils.

IX. CHROMOSOMAL MAPPING The chemokine gene CX3C has been mapped to human chromosome 16. A hybrid panel of mouse somatic cells from BIOS laboratories Laboratories (New Have, CT) was combined with PCR. These mapping studies also indicate the possibility of a pseudogene or gene related to human chromosome 14. The sequencing of the genomic DNA fragments suggests that the chemokine gene CX3C has an intron that starts near or in the codon encoding lie 64. Other limits of the intron / exon have yet to be mapped. This location is different from the chromosomal mapping locations of the other C, CC or CXC chemokine families, consistent with CX3Ccina, which is a family of genes separated within chemokines.

X. DIRECT OR INDIRECT BIOLOGICAL ACTIVITIES The 293 human embryonic kidney cell line (HEK 293) was transfected with the membrane binding form of the human CX3 293-CX3Cine, the chemokine domain plus the "stem" region, or a control vector without an insert. Subsequently transfected cells were cultured with monocytes, T cells or peripheral mono nuclear cells (PMN) to assess the relative adherence of these cells to CX3Cine. Specifically, 5 x 104 cells per well of transfected HEK 293 cells were seeded in a 96-well plate. 2 x 105 monocytes, T, or PMN, metabolically labeled with 35 S-methionia and cysteine (Amersham, Arlington Heights, IL), were added to each well. Afterwards, the plate was incubated at 37 ° C for different time points. The wells were washed twice RPMI supplemented with 1% FCS. The plates were then read in a Millipore Cytofluor at 485/530 nm.

In all cases, adhesion to HEK 293 cells transfected with the membrane-binding form of CX3Cycin was significantly improved when compared to CX3 truncated or simulated transfected cells. It is interesting to note that only the membrane-bound form exhibited this pro-adhesive activity, giving rise to the conclusion that CX3Ccina, in its membrane-bound form, can serve as a regulator of circulating leukocytes. In another experiment, the recombinant soluble form of the chemokine domain of CX3Cine (rCx3C) was added to HEK 293-CX3C cells and monocytes at a concentration of one μM per well, and taught as described above. RCx3C was able to antagonize the adhesion of monocytes to HEK 293-CX3C cells. A similar experiment was conducted to investigate the effect on the adhesion of T cells. Comparable results were obtained. In this way, rCx3C can function as a negative regulator of circulating leukocytes. A comparison of the three different forms of Human CX3C was performed to analyze variations in chemoattractant activity that may be due to the structure of CX3Ccina. The CX3C 1.7 (chemokine domain plus the entire stem region), CX3C 0.7 (chemokine domain plus half of the stem region) and CX3C CK (only the chemokine domain) were subjected to the chemotaxicity test described above, its ability to attract T cells was analyzed. The CX3C 1.7 showed a slightly better dose-pending ability to attract T cells relative to the other forms of CX3Cine. A vigorous and sensitive assay is selected as described above, for example, on immune cells, neuronal cells or germ cells. The chemokine is added to the assay in increasing doses to see if a dose response is detected. For example, in a proliferation assay the cells are plated on plates. The appropriate culture medium is provided and the chemokine is added to the cells in varying amounts. The growth is monitored over a period of time which will detect a direct effect on the cells or an indirect effect of the chemokine. Otherwise, an activation test or an attraction test is used. A suitable cell type is selected, for example, hematopoietic cells, myeloid cells (macrophages, neutrophils, polymorphonuclear cells, etc.) or lymphoid cells (T, B cells or NK cells), neural cells (neurons, neuroglia, oligodendrocytes, astrocytes, etc.), or germ cells, for example, progenitor cells that differ from other types of cells, for example, gut crypt cells and undifferentiated cell types. Other trials will be those that have been demonstrated with other chemokines. See, for example, Chali and Bacon (1994) Current Opinion in Immunology &; Immunol. 109: 97-109. The effects of truncated stem structures will be evaluated in the same way.

XI. RELATIONSHIP OF THE ACTIVITY STRUCTURE Information on the critical character of the specific residues is determined using normal procedures and analyzes. Normal mutagenesis analysis is carried out, for example, generating very different variants in determined positions, for example, in the positions as identified in the above, and evaluating the biological activities of the variants. This can be done to the extent of determining the positions that modify the activity, or to locate the specific positions in order to determine the residues that can be substituted to retain, block or modulate the biological activity. Otherwise, the analysis of natural variants may indicate which positions tolerate natural mutations. This may result from population analysis of variation between individuals, or through strains or species. Samples from selected individuals are analyzed, for example, by RCT analysis and sequencing. This allows the evaluation of population polymorphisms. Specifically, as already described, many of the biological activities of the chemokine domain bound to different portions or extensions of the stem structure can result.

XII. SIZING FOR AGONISTS / ANTAGONISTS Agonists or antagonists are screened for their ability to induce or block biological activity. The candidate compounds, for example, sequence variants of CX3 Natural cysts are tested for their biological activities. Otherwise, the compounds are screened alone or in combinations to determine effects on biological activity.

XIII. ISOLATION OF A RECEIVER FOR CX3C CHEMOCCIN Based on the pro-adherent properties of CX3Ccina, the transmembrane protein g receptor 7 was found to be expressed by monocytes and T cells. It was also discovered that the chemokine domain is the only region of the CX3. that can be coupled to the receiver. The binding assays with the known chemokine receptor showed that CX3Ccina does not bind to the chemokine receptors CCR1 to 5, CXCR1 and 2 or the Duffy antigen receptor. The CX3Cine can, however, bind to a virally encoded chemokine receptor, CMV-US28. Otherwise, the CX3C chemokine can be used, as a specific binding reagent or used against its binding part, taking advantage of its binding specificity, it would be used as an antibody. A binding reagent is labeled as described above, for example, by fluorescence or, otherwise, immobilized to a substrate for panning methods. The common chemokine receptor is a trans membrane receptor 7. The purified protein is also used to identify other binding sites of CX3Cine as described, for example, Fields and Song (1989) Nature 340: 245-246. The binding composition, eg, chemokine, is used to screen an expression library made from a cell line that expresses a binding counterpart, ie, a receptor. Normal staining techniques are used to detect or classify the expressed intracellular or surface receptor, or the transformed cells that express on the surface are sifted by panning. The screening of the intracellular expression is carried out by various staining or immunofluorescence procedures. See also McMahan et al. (1991) EMBO J. 10: 2821-2832. For example, on day 0, permanox slides are precoated with two chambers with 1 ml per fibronectin chamber, 10 ng / ml in PBS, for 30 minutes at room temperature. Rinse once with PBS. Then COS cells are plated at 2-3 x 10 5 cells per chamber in 1.5 ml of growth medium. It is incubated overnight at 37 ° C. On day 1 of each sample, 0.5 ml of a solution of 66 μg / ml DEAE-dextran is prepared, 66 μM chloroquine and 4 μg serum free DME DNA. For each series a positive control is prepared, for example, of human CX3C chemokine cDNA with a dilution of 1/200, and a negative simulated sample. The cells are rinsed with serum-free DME. The DNA solution is added and incubated for 5 hours at 37 ° C. The medium is removed and 0.5 ml of 10% DMSO DME is added for 2.5 min. Remove and rinse once with DME. Add 1.5 ml of growth medium and incubate overnight. On day 2 the medium is changed. On days three or four the cells are fixed and stained. The cells are rinsed twice with Hank's buffered saline solution (HBSS) and fixed in 4% paraformaldehyde (PFA) / glucose for 5 min. Wash 3x with HBSS. The porta porta objects can be stored at -80 ° C after all the liquid has been removed. For each chamber incubations of 0.5 ml are made as follows. Add HBSS / saponin (0.1%) with 32 μl / ml of 1M NaN3 for 20 minutes. The cells are then washed with HBSS / saponin lx. Chemokine or chemokine / antibody complex is added to the cells and incubated for 30 min. The cells are washed twice with HBSS / saponin. If appropriate, the first antibody is added for 30 min. The second antibody is added, for example, anti-mouse vector antibody, at a dilution of 1/200, and incubated for 30 min. The ELISA solution is prepared, for example, Vector Elite ABC ABC peroxidase solution and incubated for 30 min. For example, one drop of solution A (avidin) and one drop of solution B (biotin) are used per 2.5 ml of HBSS / saponin. The cells are washed twice with HBSS / saponin. The ABS HRP solution is added and incubated for 30 min. The cells are washed twice with HBSS, washed a second time for 2 min, whereupon the cells are closed. The vector diamino benzoic acid (DAB) is then added for 5 to 10 minutes. Two drops of buffer solution plus 4 drops of DAB plus two drops of H202 are used per 5 ml of vitreous distilled water. The chamber is carefully removed and the slide is rinsed in water. It is dried in air for a few minutes, then a drop of Crystal Mount is added and a cover is slipped. Bake for 5 min at 85-90 ° C.

The positive staining of the combinations is evaluated and progressively subcloned to isolate the individual genes responsible for the binding. Otherwise, chemokine reagents are used for affinity purification or classification of cells expressing a receptor. See, for example, Sanbrook et al. o Ausubel, et al. Another strategy is to sift for a receiver attached to membranes by panning. The receptor cDNA is constructed as described above. The ligand can be immobilized and used to immobilize expressing cells. Immobilization can be carried out by the use of suitable antibodies that recognize, for example, a FLAG sequence of a chemokine fusion construct or by the use of antibodies developed against the first antibodies. The recursive cycles of selection and amplification give rise to the enrichment of suitable clones and the final isolation of the clones expressing the receptor. Phage display libraries can be given by chemokine. Suitable labeling techniques, eg, anti-FLAG antibodies, will allow specific labeling of suitable clones. All references mentioned herein are incorporated by reference in the same degree as if each publication or individual patent application was specifically and individually indicated as incorporated as a reference in its integrity for all purposes. Multiple modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is limited only in the terms of the appended claims, along with the full scope of the equivalents for which these claims are entitled.

LIST OF SEQUENCES GENERAL NFORMATION: (i) APPLICANT: Schering Corporation (ii) TITLE OF THE INVENTION: CX3C MAMMERIC CHEMOCOIN GENES (iii) NUMBER OF SEQUENCES: 11 (iv) POSTAL ADDRESS: (A) RECIPIENT: Schering Corporation (B) ) STREET: 2000 Galloping Hill Road (C) CITY: Kenilworth (D) STATE: New Jersey (E) COUNTRY: EU (F) POSTAL CODE: 07033 (v) LEGIBLE COMPUTATION FORM: (A) TYPE OF MEDIA: Floppy Disk (B) COMPUTER: IBM Compatible with PC. (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Version 1.0, version 1.30 (vi) DATA OF THE CURRENT APPLICATION: (A) NUMBER OF APPLICATION: (B) DATE OF SUBMISSION: (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) NUMBER OF APPLICATION: US 08 / 649,006 (B) DATE OF SUBMISSION: May 16, 1996 (vii) DATA FROM THE PREVIOUS APPLICATION: (A) NUMBER OF APPLICATION: US 08 / 590,828 (B) DATE OF SUBMISSION: January 24, 1996 (viii) INFORMATION OF AGENT / ATTORNEY: (A) NAME: Cynthia L. Foulke, Esq. (B) REGISTRATION NUMBER: 32,364 (C) NUMBER OF REFERENCE / RECORD: DX0569K2 (ix) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: 908-298-2987 (B) TELFAX: 908-298-5388 (2) INFORMATION SEC SEC. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 534 base pairs (B) TYPE: nucleic acid (C) HEBRA: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) PECULIARITY : (A) NAME / KEY: CDS (B) LOCATION: 89,424 (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 1 (Xi) SEQUENCE descrtption: SEQ ID NO: l: CCCAAGCTTG GCACGAGGGC ACTGAGCTCT GCCGCCTGGC TCTAGCCGCC TGCCTGGCCC 60 CCGCCGGGAC TCTTGCCCAC CCTCAGCCAT GGCTCCGATA TCTCTGTCGT GGCTGCTCCG 120 CTTGGCCACC TTCTGCCATC TGACTGTCCT GCTGGCTGGA CAGCACCACG GTGTGACGAA 180 ATGCAACATC ACGTGCAGCA AGATGACATC AAAGATACCT GTAGCTTTGC TCATCCACTA 240 TCAACAGAAC CAGGCATCAT GCGGCAAACG • CGCAATCATC TTGGAGACGA GACAGCACAG 300 GCTGTTCTGT GCCGACCCGA AGGAGCAATG GGTCAAGGAC GCGATGCAGC ATCTGGACCG 360 CCAGGCTGCT GCCCTAACTC CGAAATGGCG GCACCTTCCG AAGAAGCCAG ATCGGCGAGG 420 TTGAAGCCCA GGACCACCCC CTGCCGCCGG GGGAAATGGA CNAGTCTGTT GGTCCCTGGA 480 ACCCCGAAAG CCCACAGGCG AAAAGCCAGT TACCCTGGAN CCGAATCCTT CTTC 534 (2) INFORMATION FOR SEC ID NO. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 111 amino acid (B) TYPE: amino acid (C) HEBRA: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 2: Met Wing Pro lie Ser Leu Ser Trp Leu Leu Arg Leu Wing Thr Phe Cys 1 5 10 15 His Leu Thr Val Leu Leu Wing Gly Gln His His Gly Val Thr Lys Cys 20 25 30 Asn. lie Thr Cys Ser Lys Met Thr Ser Lys lie Pro Val Wing Leu Leu 35 40 45 lie His Tyr Gln Gln Asn Gln Wing Cys Gly Lys Arg Ala lie lie 50 55 60 Leu Glu Thr Arg Gln His Arg Leu Phe Cys Wing Asp Pro Lys Glu Gln 65 70 75 80 Trp Val Lys Asp Wing Met Gln His Leu Asp Arg Gln Wing Wing Wing Leu 85 90 95 Thr Pro Lys Trp Arg His Leu Pro Lys Lys Pro Asp Arg Arg. Gly 100 105 110 (2) INFORMATION OF SEC ID NO. 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1624 base pairs (B) TYPE: nucleic acid (C) HEBRA: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) PECULIARITY : (A) NAME / KEY: CDS (B) LOCATION: 86..1276 (xi) DESCRIPTION OF SEQUENCE ID SEC NO. 3: AAGCTTGGCA CGAGGGCACT GAGCTCTGCC GCCTGGCTCT AGCCGCCTGC CTGGCCCCCG 60 CCGGGACTCT TGCCCACCCT CAGCCATGGC TCCGATATCT CTGTCGTGGC TGCTCCGCTT 120 GGCCACCTTC TGCCATCTGA CTGTCCTGCT GGCTGGACAG CACCACGGTG TGACGAAATG 180 CAACATCACG TGCAGCAAGA TGACATCAAA GATACCTGTA GCTTTGCTCA TCCACTATCA 2 0 ACAGAACCAG GCATCATGCG GCAAACGCGC AATCATCTTG GAGACGAGAC AGCACAGGCT 300 GTTCTGTGCC GACCCGAAGG AGCAATGGGT CAAGGACGCG ATGCAGCATC TGGACCGCCA 360 GGCTGCTGCC CTAACTCGAA ATGGCGGCAC CTTCGAGAAG CAGATCGGCG AGGTGAAGCC 42C CAGGACCACC CCTGCCGCCG GGGGAATGGA CGAGTCTGTG GTCCTGGAGC CCGAAGCCAC 480 AGGCGAAAGC AGTAGCCTGG AGCCGACTCC TTCTTCCCAG GAAGCACAGA GGGCCCTGGG 540 GACCTCCCCA GAGCTGCCGA CGGGCGTGAC TGGTTCCTCA GGGACCAGGC TCCCCCCGAC 600 GCCAAAGGCT CAGGATGGAG GGCCTGTGGG CACGGAGCTT TTCCGAGTGC CTCCCGTCTC 660 CACTGCCGCC ACGTGGCAGA GTTCTGCTCC CCACCAACCT GGGCCCAGCC TCTGGGCTGA 720 GGCAAAGACC TCTGAGGCCC CGTCCACCCA GGACCCCTCC ACCCAGGCCT CCACTGCGTC 780 CTCCCCAGCC CCAGAGGAGA ATGCTCCGTC TGAAGGCCAG CGTGTGTGGG GTCAGGGACA 840 GAGCCCCAGG CCAGAGAACT CTCTGGAGCG GGAGGAGATG GGTCCCGTGC CAGCGCACAC 900 GGATGCCTTC CAGGACTGGG GGCCTGGCAG CATGGCCCAC GTCTCTGTGG TCCCTGTCTC 960 CTCAGAAGGG ACCCCCAGCA GGGAGCCAGT GGCTTCAGGC AGCTGGACCC CTAAGGCTGA 1020 GGAACCCATC CATGCCACCA TGGACCCCCA GAGGCTGGGC GTCCTTATCA CTCCTGTCCC 1080 TGACGCCCAG GCTGCCACCC GGAGGCAGGC GGTGGGGCTG CTGGCCTTCC TTGGCCTCCT 1140 CTTCTGCCTG GGGGTGGCCA TGTTCACCTA CCAGAGCCTC CAGGGCTGCC CTCGAAAGAT 1200 GGCAGGAGAG ATGGCGGAGG GCCTTCGCTA CATCCCCCGG AGCTGTGGTA GTAATTCATA 1260 TGTCCTGGTG CCCGTGTGAA CTCCTCTGGC CTGTGTCTAG TTGTTTGATT CAGACAGCTG 1320 CCTGGGATCC CTCATCCTCA TACCCACCCC CACCCAAGGG CCTGGCCTGA GCTGGGATGA 1380 TTGGAGGGGG GAGGTGGGAT CCTCCAGGTG CACAAGCTCC AAGCTCCCAG GCATTCCCCA 1440 GGAGGCCAGC CTTGACCATT CTCCACCTTC CAGGGACAGA GGGGGTGGCC TCCCAACTCA 1500 CCCCAGCCCC AAAACTCTCC TCTGCTGCTG GCTGGTTAGA GGTTCCCTTT GACGCCATCC 1560 CAGCCCCAAT GAACAATTAT TTATTAAATG CCCAGCCCCT TCTGAAAAAA AAAAAAAAAA 1620 AAAAAAAAAA AAAAAAAAAA ATTCCTGCGG CCGC (2) SEC ID NO. 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 397 amino acids (B) TYPE: amino acid (C) HEBRA: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 4 Met Ala Pro lie Ser Leu Ser Trp Leu Leu Arg Leu Ala Thr Phe Cys 1 5 10 15 His Leu Thr Val Leu Leu Wing Gly Gln His His Gly Val Thr Lys Cys 20 25 30 Asn He Thr Cys Ser Lys Met Thr Ser Lye He Pro Val Wing Leu Leu 35 40 45 He His Tyr Gln Gln Asn Gln Wing Ser Cys Gly Lys Arg Ala He He 50 55 60 Leu Glu Thr Arg Gln His Arg Leu Phe Cys Wing Asp Pro Lys Glu Gln 65 70 75 80 Trp Val Lys Asp Ala Met Gln His Leu Asp Arg Gln Ala Ala Ala Leu 85 90 95 Thr Arg Asn Gly Gly Thr Phe Glu Lys Gln He Gly Glu Val Lys Pro 100 105 110 Arg Thr Thr Pro Wing Wing Gly Gly Met Asp Glu Ser Val Val Leu Glu 115 120 125 Pro Glu Wing Thr Gly Glu Ser Ser Leu Glu Pro Thr Pro Ser Ser 130 135 140 Gln Glu Ala Gln Arg Ala Leu Gly Thr Ser Pro Glu Leu Pro Thr Gly 145 150 155 160 Val Thr Gly Be Ser Gly Thr Arg Leu Pro Pro Thr Pro Lys Wing Gln 155 170 175 Asp Gly Gly Pro Val Gly Thr Glu Leu Phe Arg Val Pro Pro Val Ser 180 185 190 Thr Wing Wing Thr Trp Gln Ser Ser Wing Pro His Gln Pro Gly Pro Ser 195 200 205 Leu Trp Wing Glu Wing Lys Thr Ser Glu Wing Pro Ser Thr Gln Asp Pro 210 215 220 Ser Thr Gln Wing Ser Thr Wing Ser Ser Wing Pro Pro Glu Glu Asn Wing 225 230 235 240 Pro Ser Glu Gly Gln Arg Val Trp Gly Gln Gly Gln Pro Pro Arg Pro 245 250 255 Glu Asn Ser Leu Glu Arg Glu Glu Met -Gly Pro Val Pro Wing His Thr 260 265 270 Asp Wing Phe Gln Asp Trp Gly Pro Gly Ser Met Wing His Val Ser Val 275 280 285 Val Pro Val Ser Ser Glu Gly Thr Pro Ser Arg Glu Pro Val Wing Ser 290 295 300 Gly Ser Trp Thr Pro Lys Wing Glu Glu Pro lie His Wing Thr Met Asp 305 310 315 320 Pro Gln Arg Leu Gly Val Leu He Thr Pro Val Pro Asp Ala Gln Wing 325 330 335 Wing Thr Arg Arg Gln Wing Val Gly Leu Leu Wing Phe Leu Gly Leu Leu 340 345 350 Phe Cys Leu Gly Val Wing Met Phe Thr Tyr Gln Ser Leu Gln Gly Cys 355 360 365 Pro Arg Lys Met Wing Gly Glu Met Wing Glu Gly Leu Arg Tyr He Pro 370 375 380 Arg Ser Cys Gly Ser Asn Ser Tyr Val Leu Val Pro Val 385 390 395 (2) INFORMATION OF SEC ID NO. 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 209 base pairs (B) TYPE: nucleic acid (C) HEBRA: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) PECULIARITY : (A) NAME / KEY: CDS (B) LOCATION: 69..209 (xi) DESCRIPTION OF SEQUENCE ID SEC NO. 5: TNACTACTAG GAGCTGCGAC ACGGCCCAGC CTCCTGGCCC GNCGAATTCC TGCACTCCAG 60 CCATGGCTCC CTCGCCGCTC GCGTGGCTGC TGCGCCTGGC CGCGTTCTTC CATTTGTGTA 120 CTCTGCTGCC GGGTNAGCAC CTCGGCATGA CGAAATGCGA AATCATGTGC GACAAGATGA 180 CCTNACGAAT NCCAGTGGCT TTANTCATC 209 (2) INFORMATION OF SEC ID NO. 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 49 amino acids (B) TYPE: amino acid (C) HEBRA: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE : SEC ID NO. 6: Met Ala Pro Ser Pro Leu Ala Trp Leu Leu Arg Leu Ala Ala Phe Phe 1 5 10 15 His Leu Cys Thr Leu Leu Pro Gly Xaa His Leu Gly Met Thr Lys Cys 20 25 30 Glu He Met Cys Asp Lys Met Thr Xaa Arg Xaa Pro Val Ala Leu Xaa 35 40 45 He (2) INFORMATION OF SEC ID NO. 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3065 base pairs (B) TYPE: nucleic acid (C) HEBRA: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) PECULIARITY : (A) NAME / KEY: CDS (B) LOCATION: 62..1249 13. (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 7: TGACTACTAG GAGCTGCGAC ACGGCCCAGC CTCCTGGCCG CCGAATTCCT GCACTCCAGC 60 C ATG GCT CCC TCG CCG CTC GCG TGG CTG CTG CGC CTG GCC GCG TTC 106 Met Wing Pro Pro Leu Wing Trp Leu Leu Arg Leu Wing Wing Phe 1 5 10 15 TTC CAT TTO TGT ACT CTG CTG CCG GGT CAG CAC CTC GGC ATG ACG AAA 154 Phe His Leu Cys Thr Leu Leu Pro Gly Gln His Leu Gly Met Thr Lye 20 25 30 TGC GAA ATC ATG TGC GGC AAG ATG ACC TCA CGA ATC CCA GTG GCT TTG 202 Cys Glu He Met Cys Gly Lys Met Thr Ser Arg He Pro Val Ala Leu 35 40 45 CTC ATC CGC TAT CAG CTA AAT CAG GAG TCC TGC GGC AAG CGT GCC ATT 250 Leu He Arg Tyr Gln Leu Asn Gln Glu Ser Cys Gly Lys Arg Ala He 50 55 60 GTC CTG GAG ACG ACÁ CAG CAC AGA CGC TTC TGT GCT CCG AAG GAG 298 Val Leu Glu Thr Thr Gln His Arg Arg Phe Cys Wing Asp Pro Lys Glu 65 70 75 AAA TGG GTC CAA GAC GCC ATG AAG CAT CTG GAT CAC CAG GCT GCT GCC 346 Lys Trp Val Gln Asp Wing Met Lys Hie Leu Asp His Gln Wing Wing Wing 80 85 90 95 CTC ACT AAA AAT GGT GGC AAG TTT GAG AAG CGG GTG GAC AAT GTG ACA 394 Leu Thr Lys Asn Gly Gly Lys Phe Glu Lys Arg Val Asp Asn Val Thr CCT GGG ATC ACC TTG GCC ACT AGG GGA CTG TCC CCA TCT GCC CTG ACA 442 Pro Gly He Thr Leu Wing Thr Arg Gly Leu Ser Pro Be Wing Leu Thr 115 120 125 AAG CCT GAA TCC GCC ACA TTG GAA GAC CTT GCT TTG GAA CTG ACT ACT 490 Lys Pro Glu Be Wing Thr Leu Glu Asp Leu Wing Leu Glu Leu Thr Thr 130 135 140 ATT TCC CAG GAG GCC AGG GGG ACC ATG GGG ACT TCC CAA GAG CCA CCG 538 He Ser Gln Glu Ala Arg Gly Thr Met Gly Thr Ser Gln Glu Pro Pro 145 150 155 GCA GCA GTG ACC GGA TCA TCT CTC TCA ACT TCC GAG GCA CAG GAT GCA 586 Wing Wing Val Thr Gly Ser Ser Leu Ser Thr Ser Glu Wing Gln Asp Wing 160 165 170 175 GGG CTT ACG GCT AAG CCT CAG AGC ATT GGA AGT TTT GAG GCG GCT 63 Gly Leu Thr Wing Lys Pro Gln Ser He Gly Ser Phe Glu Wing Wing Asp 180 185 190 ATC TCC ACC ACC GTT TGG CCG AGT CCT GCT GTC TAC CA TCT GGA TCT 6S2 Be Ser Thr Thr Val Trp Pro Ser Wing Val Tyr Gln Ser Gly Ser 195 200 205 AGC TCC TGG GCT GAA AAA GCT ACT GAG TCC CCC TCC ACT ACA GCC 730 Ser Ser Trp Wing Glu Glu Lys Wing Thr Glu Ser Pro Ser Thr Thr Wing 210 215 220 CCA TCT CCT CAG GTG TCC ACT ACT TCA CCT TCA ACC CCA GAG GAA AAT 7 8 Pro Ser Pro Gln Val Ser Thr Thr Ser Pro Ser Thr Pro Glu Glu Asn 225 230 235 GTT sss TCC GAA GGC CAÁ CCC CCA TGG GTC CAG GGA CAG GAC CTC AGT 826 Val Gly Ser Glu Gly Gln Pro Pro Trp Val Gln Gly Gln Asp Leu Ser 240 245 250 255 CCA GAG AAG TCT CTA GGG TCT GAG GAG ATA AAC CCA GTT CAT ACT GAT 874 Pro Glu Lys Ser Leu Gly Ser Glu Glu As Asn Pro Val His Thr Asp 260 265 270 AAT TTC CAG GAG AGG GGG CCT GGC AAC ACA GTC CAC CCC TCA GTG GCT 922 Asn Phe Gn Glu Arg Gly Pro Gly Asn Thr Val His Pro Ser Val Wing 275 280 285 CCC ATC TCC TCT GAA GAG ACC CCC AGC CCA GAG CTG GTG GCC TCG GGC 970 Pro He Ser Ser Glu Glu Thr Pro Ser Pro Glu Leu Val Wing Ser Gly 290 295 300 AGC CAG GCT CCT AAG ATA GAG GAA CCC ATC CAT G CC ACT GCA GAT CCC 1018 Be Gln Wing Pro Lys He Glu Glu Pro He His Wing Thr Wing Asp Pro 305 310 315 CAG AAA CTG AGT GTG CTT ATC ACT CCT GTC CCC GAC ACC CAG GCA GCC 1066 Gln Lys Leu Ser Val Leu He Thr Pro Val Pro Asp Thr Gln Wing Wing 320 325 330 335 ACÁ AGG AGG CAG GCA GTG GGG CTA CTG GCT TTC CTT GGT CTT CTT TTC 1114 Thr Arg Arg Gln Wing Val Gly Leu Leu Wing Phe Leu Gly Leu Leu Phe 340 345 350 TGC CTA GGG GTG GCC ATG TTT GCT TAC CAG AGC CTT CAG GGC TGT CCC 1162 Cys Leu Gly Val Wing Met Phe Wing Tyr Gln Ser Leu Gln Gly Cys Pro CGC AAA ATG GCG GGG GAA ATG GTA GAA GGC CTC CGC TAC GTC CCC CGT 1210 Arg Lys Met Wing Gly Glu Met Val Glu Gly Leu Arg Tyr Val Pro Arg 370 375 380 AGC TGT GGC AGT AAC TCA TAC GTC CTG GTG CCA GTG TGA GCTGCTTGCC 1259 Ser Cys Gly Ser Asn Ser Tyr Val Leu Val Pro Val * 385 390 395 TGCCTGCCTG TGTCCAGAOT GTGATTCGGA CAGCTGTCTG GGGACCCCCC CCCATCCTCA 1319 TACCCACCTT CATCCACGCT GGGGAAATGG GAATGGAGAA GCTGGACCTC CAGGGGCTGT 1379 GGGCTCCATC CAATCCCCCT TCCCCCGAGG GGTGGCCCCG GAGGCCACCC TAGACCACTA 1439 TTCACTTATC AGAGACAGAG CAGGTGACCT TCCAGCTCCT CTATATTTGA AAGAATCCTC 1499 TGCTGCTGGC TGGTTAGAGG GGCCCTTGAC ACCCCAACTC CAGTGAACAA TTATTTATTG 1559 GATTCCCAGC CCCTGCGACG ACACCTGTTT CCCGCGCGCA CCGTGGTCCG CCCATATCAC 1619 AAGCAGCAGG CCAGGCCTAT CTGCCTGTCC CCCTGACCTC CTTGTGTCTC CTGGCTTTGC 1679 TGCAGTCGCC AGCCCTTCTC CTCCCCGGCC AGCCGCGGTG CTATCTGCCC TATGTCTCCC 1739 TCTATCCCCT GTACAGAGCG CACCACCATC ACCATCAACA CCGCTGTTGT GTCTTTTCTT 1799 GCATGAGGTT AAAGCTGTGT TTTCTGGAGC TCTCCGGGAA GGGAGACAAG CTTGCGAGAG 1859 GGTTTAAAGT GTTCCTCCCC AGAC TGGAT GTGCTGTGAG GGCATGCTGC GTCTGAAGGA 1919 AGGGTCCAGT CCCCACTCGG CTACCAGCAC CACAAAGTGC CCCACCTGTA AAAGGAAAGA 1979 AACGTGGTCC AGAGCTGGCA ATAACCTATG GCCCTGACAT CATCACTTTC TCTGAGATCC 2039 TTGTCTCCAC CCCTGGGTGC AACCCCACCC CTTATCAACA TTAATAGTCA CTGCCATTCC 099 ACTGGACTGA CATTTTTGTA CCCTGTGATT CTGAGGGCTG GCAAGGAGTG GCTTGAGAGT 21-59 GCAGATCGTA CCCTGTATGC CCCCCCCAAA TGGAGGCTGA GTTGGGGACT TGCAGGAACA 2219 GAGGCCAACT CAGATGGCTT CCCCTGTGTT CTCACTAGAA ACCCCTCCCC CATGCACCAA 2279 GGTGACAGTC ACAGGTCTGC CCTGGCTAAA GGACAAGCCA CATAGGAAAG ATTAGGACAA 2339 GCCCCTCGGA GGCAGAGGAT CCAGGsTAAA CCCCTGGAGT GGCCACAAAC CCAATTTCAG 2399 TGTAGGGACT TGTGCATGTG TGTACTTGCA TAGTCAGACA GAGGCTGCCA GGGTCCTTTC 2459 CTGTCTCTGA GAGCAGTGTT CACGCCAAGG ACTCACCTTT GCCCCCATTG CAGGCAGGGC 2519 CAGAACTCCC ATAGCATTCT CCAAGAGCCC TGTGACATTT TCTGGAAGGA ACTCTGCCCT 2579 GGGCGCAAAG TGACTGCTGA AGCAAGGAGC AGCTGAGCAG CACCCCAGCG GAGCTGAGCC 2639 GGCAGGCCAC GCCCCTCGGG GGGGGGCATT TCTACCCGCC CTGCTCTGAA TAGCTCCAAC 2699 TTCACCTTAG GAGCCTCCCA GGGGCGAGCT TCACCCAGAA GCCAGTGACT CACTCCTTGA 2759 TTGGTGGAAG CTCAGTTGGC TCCTGAGAGT GAGGAAGCCA ACCCTTTGTC GACCCTCCTC 2819 CTGGGAAGCC TGTGGGCGGC TCTGATCATG CTCCACAGAA CCAGTTGTAG GCCTGAGCCG 2879 CAGCAGCCCG AGTGCACTAT ATCCTGGCTC CTTCGGTGGG GAACCTTTAA GGGTTGGGAC 2939 ACCCGTCATC GGACTTTGTT GGTTCCTCCC TCCCAGAGCA GAATGTGGGC CGTAACAATC 2999 TGAGGAGGAC TTTAAAAGTT GTTGATCCTT TAGGGTTTTT TTTCAAGCAT CATTACCAAT 3059 GTCTGT 3065 ) INFORMATION OF SEC ID NO. 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 396 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE ID SEC NO. 8: Met Ala Pro Ser Pro Leu Ala Trp Leu Leu Arg Leu Ala Ala Phe Phe 1 5 10 15 His Leu Cys Thr Leu Leu Pro Gly Gln His Leu Gly Met Thr Lys Cys 20 25 30 Glu He Met Cys Gly Lys Met Thr Ser Arg He Pro Val Ala Leu Leu 35 40 45 He Arg Tyr Gln Leu Asn Gln Glu Ser Cys Gly Lys Arg Ala He Val 50 55 60 Leu Glu Thr Thr Gln His Arg Arg Phe Cys Ala A = p Pro Lys Glu Lys 65 70 75 80 Trp Val Gln Asp Ala Met Lys His Leu Asp His Gln Ala Ala Ala Leu 85 90 95 Thr Lys Asn Gly Gly Lys Phe Glu Lys Arg Val Asp Asn Asn Val Thr Pro 100 105 110 Gly He Thr Leu Wing Thr Arg Gly Leu Ser Pro Wing Leu Thr Lys 115 120 125 Pro Glu Be Wing Thr Leu Glu Asp Leu Wing Leu Glu Leu Thr Thr He 130 135 140 Ser Gln Glu Wing Arg Gly Thr Met Gly Thr Ser Gln Glu Pro Pro Wing 145 150 155 160 Wing Val Thr Gly Ser Ser Leu Ser Thr Ser Glu Wing Gln Asp Wing Gly 165 170 175 Leu Thr Wing Lys Pro Gln Ser He Gly Ser Phe Glu Wing Wing Asp He 180 185 190 Ser Thr Thr Val Trp Pro Ser Wing Val Tyr Gln Ser Gly Ser Ser 195 200 205 Ser Trp Wing Glu Glu Lys Wing Thr Glu Ser Pro Thr Thr Pro Wing 210 215 220 Pro Pro Gln Val Ser Thr Thr Ser Pro Ser Thr Pro Glu Glu Asn Val 225 230 235 240 Gly Ser Glu Gly Gln Pro Pro Trp Val Gln Gly Gln Asp Leu Ser Pro 245 250 255 Glu Lys Ser Leu Gly Ser Glu Glu He Asn Pro Val His Thr Asp Asn 260 265 270 Phe Gln Glu Arg Gly Pro Gly Asn Thr Val His Pro Ser Val Wing Pro 275 280 285 He Ser Ser Glu Glu Thr Pro Ser Pro Glu Leu Val Wing Ser Gly Ser 290 295 300 Gln Wing Pro Lys He Glu Glu Pro He His Wing Thr Wing Asp Pro Gln 305 310 315 320 Lys Leu Ser Val Leu He Thr Pro Val Pro Asp Thr Gln Ala Wing Thr 325 330 335 Arg Arg Gln Ala Val Gly Leu Leu Ala Phe Leu Gly Leu Leu Phe Cys 340 345 350 Leu Gly Val Ala Met Phe Ala Tyr Gln Ser Leu Gln Gly Cys Pro Arg 355 360 365 Lys Met Wing Gly Glu Met Val Glu Gly Leu Arg Tyr Val Pro Arg Ser 370 375 380 Cys Gly Ser Asn Ser Tyr Val Leu Val Pro Val * 385 390 395 (2) INFORMATION FOR SEC ID NO. 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 96 amino acids (B) TYPE: amino acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xii) DESCRIPTION OF THE SEQUENCE : SEC ID NO. 9: Met He Pro Ala Thr Arg Ser Leu Leu Cys Ala Ala Leu Leu Leu Leu 1 5 10 15 Wing Thr Ser Arg Leu Wing Thr Gly Wing Pro Wing Wing Asn Glu Leu Arg 20 25 30 Cys Gln Cys Leu Gln Thr Met Wing Gly He His Leu Lys Asn He Gln 35 40 45 Ser Leu Lys Val Leu Pro Ser Gly Pro His Cys Thr Gln Thr Glu Val 50 55 60 He Wing Thr Leu Lys Asn Gly Arg Glu Wing Cys Leu Asp Pro Glu Wing 65 70 75 80 Pro Leu Val Gln Lys He Val Gln Lys Met Leu Lys Gly Val Pro Lys 85 90 95 (2) INFORMATION FOR SEC ID NO. 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 115 amino acids (B) TYPE: amino acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE : SEC ID NO. 10: Met Arg Leu Leu Leu Leu Thr Phe Leu Gly Val Cys Cys Leu Thr Pro 1 5 10 15 Tr Val Val Glu Gly Val Gly Thr Glu Val Leu Glu Glu Ser Ser Cys 20 25 '30 Val Asn Leu Gln Thr Gln Arg Leu Pro Val Gln Lys He Lys Thr Tyr 35 40 45 He He Trp Glu Gly Wing Met Arg Ala Val He Phe Val Thr Lys Arg 50 55 60 Gly Leu Lys He Cys Wing Asp Pro Glu Wing Lys Trp Val Leu Wing Wing 65 70 75 80 He Lys Thr Val Asp Gly Arg Wing Being Thr Arg Lys Asn Met Wing Glu 85 90 95 Thr Val Pro Gly Thr Gly Wing Gln Arg Ser Thr Ser Thr Wing He Thr 100 105 110 Leu Thr Gly 115 (2) INFORMATION FOR SEC ID NO. 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 92 amino acids (B) TYPE: amino acid (C) HEBRA: simple (D) TOPOLOGY: linear (Ü) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. eleven Met Lys Leu Cys Val Ser Ala Leu Ser Leu Leu Leu Leu Val Ala Ala 1 5 10 15 Phe Cys Wing Pro Gly Phe Be Wing Pro Met Gly Ser Asp 'Pro Pro Thr 20 25 30 Ser Cys Cys Phe Ser Tyr Thr Wing Arg Lys Leu Pro Arg Asn Phe Val 35 40 45 Val Asp Tyr Tyr Glu Thr Ser Ser Leu Cys Ser Gln Pro Wing Val Val 50 55 60 Phe Gln Thr Lys Arg Ser Lys Gln Val Cys Wing Asp Pro Ser Glu Ser 65 70 75 80 Trp Val Gln Glu Tyr Val T r Asp Leu Glu Leu Asn 85 90

Claims (20)

1. An antibody binding site that specifically binds to a mammalian C43C [sic] chemokine.

2. The antibody binding site of claim 1 which is specifically immunoreactive with a protein having the amino acid sequence set forth in SEQ ID NO. 2, 4, 6 or 8.

3. The antibody binding site of claim 1, which is found in a Fab or F (ab) 2 monoclonal antibody.

4. The antibody binding site of claim 1, which is in a labeled antibody.

5. The antibody binding site of claim 1, which is developed against a human or purified CX3C chemokine or recombinantly produced mouse.

6. A substantially pure protein that is recognized by the antibody binding site of claim 1.

7. A method for detecting the antibody binding site of claim 1 in a biological sample, which consists of the steps of: a) contacting a binding agent having an affinity to the CX3C chemokine protein with the biological sample; b) incubating the binding agent with the biological sample to form a binding agent: the chemokine protein complex CX3C; c) detect the complex.

The method of claim 7, wherein the biological sample is human, and wherein the binding agent is an antibody.

9. An expression vector consisting of nucleic acid encoding a mammalian CX3C chemokine or fragment thereof.

The vector of claim 9, which encodes a CX3C chemokine protein, wherein the protein specifically binds to an antibody generated against an immunogen that is selected from the group consisting of the polypeptide of SEQ ID NO: 2, 4 , 6 and 8.

The vector of claim 9, which: a) encodes a CX3C chemokine polypeptide with complete sequence identity for a human CX3C chemokine protein in its native state; b) encodes a CX3C chemokine protein containing the sequence selected from the group consisting of the polypeptide of SEQ ID NO. 2, 4, 6 and 8; or c) comprises a sequence selected from the group consisting of the nucleic acid of SEQ ID NO. 1, 3, 5 and 7.

12. The vector of claim 9, which is capable of selectively hybridizing to a nucleic acid encoding a CX3C chemokine protein.

13. The vector of claim 9, which comprises a mature protein encoding the segment of SEQ ID NO. 1, 3, 5 o7.

14. A cell transfected with the vector of claim 9.

15. The cell of claim 14, wherein the nucleic acid consists of a polynucleotide sequence selected from the group consisting of the nucleic acid of SEQ ID NO: 1, 3, 5 and 7.

16. A substantially pure CX3C chemokine or peptide fragment thereof, or a fusion protein thereof.

17. The CX3C chemokine protein of claim 16, wherein the CX3C chemokine protein: a) is produced recombinantly, or b) is a protein in its native state.

18. The protein of claim 16, wherein the CX3C chemokine protein: a) is selected from the group consisting of CX3 human CX3 and CX3 mouse; or b) consists of a polypeptide that is selected from the group consisting of SEQ ID NO: 2, 4, 6 and 8.

19. A CX3C chemokine protein isolated from about 11,000 to 12,500 daltons when in its non-glycosylated form, wherein the chemokine protein CX3C binds specifically to an antibody raised against an immunogen selected from the group consisting of: a) the ID polypeptide SEQ NO: 2; b) the polypeptide of SEQ ID NO: 4; c) the polypeptide of SEQ ID NO: 6; and d) the polypeptide of SEQ ID NO: 8. and the CX3C chemokine lack the structural motifs of cysteine and the characteristic sequence of a C, CC or CXC chemokine.

20. A method of modulating the physiology or development of a cell comprising: contacting the cell with a CX3C chemokine or a chemokine antagonist.