MXPA98005598A - Exodus chemokine materials and methods - Google Patents

Abstract

The present invention provides purified and isolated polynucleotide sequences encoding a human macrophage-derived C-C chemokine designated Exodus. Also provided are purified and isolated chemokine protein, fragments and polypeptide analogs thereof, antibodies thereto, and materials and methods for the recombinant production thereof. These products are useful in therapeutic, diagnostic and medical imaging applications.

Description

- 3-W "EXODUS CHEMIOCIN MATERIALS AND METHODS" The present invention relates generally to chemokines and more particularly to purified and isolated polynucleotides encoding a human C-C chemokine designated Exodus and the analogs thereof with purified and isolated chemokine polypeptides encoded by the polynucleotides, with materials and * methods for the recombinant production of these 10 polypeptides and therapeutic uses of these polypeptides, particularly in myeloprotection during chemotherapy, in the treatment of myeloproliferative diseases, and for the acquired immunodeficiency syndrome (AIDS). BACKGROUND OF THE INVENTION The chemokines also known as "intercrine" and "SIS cytokines" comprise a superfamily of small secreted proteins (approximately 70 to 100 amino acids and 8 to 12 kilodaltons in size) that mainly regulate the migration and activation of leukocytes and in this way help the stimulation and regulation of the immune system. Name "chemokine" is derived from the term chemotactic cytokine, # and refers to the ability of these proteins to stimulate the chemotaxis of leukocytes. Of course, chemokines can comprise the main attraction materials for inflammatory cells towards pathological tissues. [See generally, Baggiolini et al., Advances in Immunology, 55: 97-179 (1994); Oppenheim, The Cehomokines, Lindley and others, editors, pages 183-86, Plenum Press, NY (1993))]. Even though chemokines are generally secreted by leukocytes, several chemokines are expressed in a multitude of tissues. Baggiolini et al., Supra, Table II. Some chemokines also activate or attract a variety of cell types in addition to leukocytes such as endothelial cells and fibroblasts. 15 Chemokines previously identified by # They generally exhibit 20 percent to 70 percent amino acid identity with respect to each other and contain four highly conserved cysteine residues. Based on the relative position of the first two of these cysteine residues, the chemokines have been further classified into two subfamilies. In the subfamily "C-X-C" or "a", encoded by genes located on human chromosome 4, the first two cysteines are separated by an amino acid. In the subfamily "C-C" or "ß", encoded by genes that have been mapped to human chromosome 17, the first two cysteines are adjacent. X-ray crystallography and nuclear magnetic resonance studies of several chemokines have indicated that in each family, the first and third cysteines form a first disulfide bridge, and the second and fourth cysteines form a second disulfide bridge, strongly influencing the active conformation of proteins. In humans alone, almost ten different sequences have been described for each chemokine subfamily. The chemokines of both subfamilies have characteristic forward sequences of twenty to twenty-five amino acids. The CXC chemokines, which include IL-8, GROa / β / ?, the basic platelet protein, Platelet Factor 4 (PF4), neutrophil activation peptide 2 (NAP-2), the chemotactic factor and Macrophage activation (MCAF), IP-10 and others, share approximately 25 percent to 60 percent identity when any two amino acid sequences are compared (except for GROa / ß /? members, which are 84 percent to 88 percent identical one with respect to the other). The majority of members of the subfamily (excluding IP-10 and Platelet Factor 4) share a tripeptide motif E-L-R common to the first two cysteine residues. C-X-C chemokines are usually "potent neutrophil stimulants, causing a field of rapid form, chemotaxis, respiratory bursts and degranulation. The specific tuning of the N-terminal amino acid sequence of certain chemokines C-X-C 5 including IL-8, is associated with marked increases in activity. C-C chemokines, which include the Proteins Inflammatory Macrophage MlP-la [Nakao et al., Mol. Cell Bil., 10: 3646 (1990)] and MlP-lß [Brown et al., J.

Im unol., 142: 679 (1989)], Monocyte Chemostactic Proteins MCP-1 [Matsushi et al., J. Exp. Med., 169: 1485 (1989)], MCP-2 [Van Damme et al., J. Exp. Med., 176: 59 (1992) and Chang et al., Int. Immunol. , 1: 388 (1989)], and MCP-3 [Van Damme et al., Supra], RANTES [Schall et al., J.

Immunol., 141: 1018 (1988)], 1-309 [Miller et al., J. Immunol., 143: 2907 (1989)] eotaxin [Rothenberg et al., J. Exp. Med., 181: 1211 -1216 (1995)] and others, share 25 percent to 70 percent of the identity of the amino acid one with respect to the other. C-C chemokines generally activate monocytes, lymphocytes, basophils and eosinophils, but not neutrophils. Most of the C-C chemokines disclosed activate monocytes, causing calcium flux and chemotaxis. The most selective effects are seen in lymphocytes, for example, T-25 lymphocytes, which respond more intensely to -RANTES.

C-C chemokines can also be subdivided according to structural homologies and similar activities. MlP-la, MlP-lß and RANTES have homology and scale of biological activities closer than the other 5 members of the family. Another subfamily within the C-C chemokine family are the onocito chemotactic proteins (MCP), which are structurally more Ofa-like than the other members of the C-C chemokine family, and which preferentially stimulate the raonocytes to develop and respond to inflammatory stimuli. Studies with deletion and substitution analogs have revealed that the critical receptor binding regions appear to remain mainly in the amino-terminal residues of the chemokines followed by -J? a second region in the circuit following the second cysteine. These general requirements for function appear to be common for all chemokines [Clark-Lewis et al., J. Leukocyte Bio., 57: 703 (1995).] 20 Chemokine receptors are seven-transmembrane receptors-rhodopsin domain resembled protein G coupled. A specific receptor for IL-8 has been cloned by Holmes et al., Science, 253: 1278-83 (1991), while a similar receptor (identity of 77 by percent) that recognizes IL-8, GRO and NAP-2 has been cloned by - Murpny and Tiffany, Science, 253: 1280-83 (1991). Five of the CC chemokine receptors have been cloned to date: a CCl chemokine receptor (CCR-1) that recognizes MlP-la and RANTES [Neote et al., Cell, 72: 415-425 (1993)], a receptor (CCR-4) for MlP-la RANTES and MCP-1 [Power et al., J. Biol. Chem., 270: 19495-19500 (1995)], and the MCP-1 receptor (CCR-2B) [Charo and others, Proc. Nat. Acad. Sci., 91: 1752-56 (1994)], an eotaxin receptor (CCR-3) [Combadiere et al., J. Biol. Chem. 270: 16491-16494 (1995)], and a Receptor (CCR-5) for MlP-la, MlP-lβ and RANTES [Raport et al., J. Biol. Chem., 271: 17161-17166 (1996)]. These receptors tend to be multifunctional, and can bind a number of different chemokines. The receptors themselves may have a role in the disease of the human being. For example, the Duffy antigen in human red blood cells (also known as erythrocyte chemokine receptor), which binds chemokines including IL-8, NAP-2, GROa, RANTES, MCP-1, is a receptor for invasion for the parasite that causes the malaria, Plasmodium knowlesi. Two herpesviridae, Herpesvirus saimiri and the human cytomegalovirus, also appear to be coding for the functional chemokine receptor homologs. [Ahuja et al., Immunol. Today, 15: 281- (1994); Murphy, Ann. Rev. Immnol., 12: 593-633 (1994); Horuk, TIPS, 15: 159 (1994).] ^ Because of its pro-inflammatory activities, chemokines are thought to play a role in a wide variety of diseases involving the destruction of inflammatory tissue such as rheumatoid arthritis, myocardial infarction and the adult respiratory distress syndrome. The role of a number of chemokines, particularly the chemokine C-X-C IL-8, has been well documented in different pathological conditions. See generally the Table ? t VII by Baggiolini et al., supra. For example, several studies have observed high levels of IL-8 in the synovial fluid of the inflammatory joints of patients suffering from rheumatic diseases, osteoarthritis and gout. Psoriasis has also been linked to the overproduction of IL-8. 15 The role of C-C chemokines in pathological conditions is also well documented. For example, the concentration of MCP-1 is higher in the synovial fluid of patients suffering from rheumatoid arthritis than that of patients suffering from other arthritic diseases. The MCP-1-dependent influx of mononuclear phagocytes may be an important event in the development of idiopathic pulmonary fibrosis. The role of C-C chemokines in the recruitment of monocytes to atherosclerotic areas at present is of interest intense with improved MCP-1 expression having been detected in the areas of the arterial wall rich in macrophages but in the normal arterial tissue. MCPs may also be involved in the induction of angiogenesis and tumor growth or metastasis. The expression MCP-1 in malignant cells has been shown to suppress the ability of these cells to form tumors in vivo. (See U.S. Patent Number 5,179,078, incorporated herein by reference). Other chemokine activities include the ability to inhibit proliferation of progenitor cells from the bone marrow. Recombinant MlP-la, but not MlP-lß, have been shown to suppress myelopoiesis of progenitor and hemocytoblast cells and appear to be selective in their ability to suppress proliferation stimulated by multipotent progenitor cell growth factor ( granulocyte-erythroid-macrophage-megakaryocyte colony-forming units, FCU-GEMM) and subpopulations of erythroid burst-forming units (BFU-E) and colony-forming units of granulocyte-macrophage progenitor cells (CFU-GM). [Broxmeyer et al., Blood, 76: 1110-1116 (1990).] These effects are not a cytotoxic effect, but rather an arrest- of the cell cycle. MIP-2a, IL-8, PF4 and MCAF have also been reported as being proliferative suppressors of "# hemopoietic hemopoietic cells / progenitor cell." [Broxmeyer et al., J. Immunol., 150: 3448-3458 (1993); Broxmeyer and others, Ann. Hematol, 71: 235-246 (1995).] These chemokines appear to act directly at the level of the myeloid progenitors. Some reports indicate that MlP-la has the potential to protect multipotent hematopoietic cells from the cytotoxic effects of ^ The chemotherapeutic agents. [Dunlop et al., Blood, i 79: 2221-2225 (1992) and Lord et al., Bloode, 79: 2605-2609 (1992).] Clinical trials are reportedly being conducted for the use of an MlP-la analogue (designated BB10010, British Biotechnology) as a myeloprotective agent with Citoxan® (Bristol-Myers Squibb Oncology cyclophosphamide). 15 Recently, there have been several reports that some chemokines C-C, MlP-la, MlP-lß and RANTES, inhibit the production of human immunodeficiency virus (HIV). [Cocchi et al., Science, 270: 1811 (1996); Fauci, Nature, 378: 561 (1996).] A study reported that CD4 + lymphocytes from people who have been exposed to HIV but remain negative to HIV express very high levels of these C-C chemokines. [Paxton et al., Nature Med., 2: 412 (1996).] A potential mechanism for this inhibition has been suggested by isolation and identification of the HIV co-receptors as members of the chemokine receptor families. The CCR-5 receptor that binds RANTES, MlP-la and MlP-lβ has been identified as the principal co-receptor for most strains of tropic-macrophage HIV [Deng et al., Nature, 381: 661 (1996); Dragic et al., Nature, 381: 667 (1996); Alkhatib et al., Science, 272: 1995 (1996). It has been reported that the occasional primary HIV-1 tropic-macrophage strains interact with CCR-3 and CCR-3 receptors in vi tro [Choe et al., Cell, 85: 1135 (1996); Doranz et al., Cell, 85: 1149 (1996)]. A chemokine receptor designated "Fusin" (now known as the chemokine receptor C-X-C CXCR-4) has been identified as a receptor for tropic HIV T-cell strains [Feng et al., Science, 272: 872 (nineteen ninety six)] . These HIV co-receptors are in the chemokine receptor families and appear to be cofactors with CD4 for the fusion and entry of HIV viruses into human target cells. Therefore there is a need for identification and characterization of additional C-C chemokines in order to further clarify the role of this important family of molecules in the pathological conditions and to develop improved treatment for these conditions using chemokine-derived products. < *. Of interest for the present invention is International Publication Number WO 96/05856 published February 29, 1996, which discloses the identification of two chemokines called human chemokine beta-4 (Ckß-4) and human chemokine beta-10 ( Ckβ-10) of cDNA libraries derived from the gallbladder and human fetal tissue for nine weeks, respectively. Ckß-4 is very similar in both the DNA and the amino acid sequence to the Exodus chemokine described in present (the differences being that Ckß-4 an additional alanine after residue 4 of the Exodus chemokine matures and that the forward deduced sequence reported from Ckß-4 is 24 amino acids, as compared to the forward Exodus sequence of 22 amino acids) . I dont know determined no biological activities neither chemokine Ckß-4 nor Ckß-10. In particular, the publication does not mention any potential role for these chemokines in the pathogenesis of HIV infection, nor does it specifically describe the use of these chemokines to treat myeloproliferative diseases. Also of interest is the cloning of another C-C chemokine designated Exodus-2, which appears to be closely related to Exodus / MIP-3a-LARC, sharing the identity of 31 percent amino acid and the same unique motif of Asp-Cys-Cys-Leu that looks around the first two cysteines. [Hromas et al., J. Immunol., 159: 2554-2558 (1997).] Chemokines of the CC subfamily have been shown to possess utility and medical imaging information, e.g., to image the site of infection , inflammation, and other sites that have CC chemokine receptor molecules. See, e.g., of Kunkel et al., U.S. Patent Number 5,413,778, which is incorporated herein by reference. These methods involve the chemical fixation of an irradiation agent (v.gr, a radioactive isotope) for C-C chemokine using recognized techniques in the field (see, v.gr, Patents North American Nos. 4,965,392 and 5,037,630, which are incorporated herein by reference), the administration of the chemokine irradiated to a patient in the pharmaceutically acceptable carrier, allowing the irradiated chemokine to accumulate in a target site, and to image the chemokine irradiated in vivo at the target site. There is a need in the art for new additional C-C chemokines to increase the available arsenal of medical imaging tools. More generally, due to the importance of chemokines as mediators of chemotaxis and inflation, there is a need for the identification, and isolation of new members of the chemokine family to facilitate the modulation of inflammatory and immune responses. For example, substances that promote the immune response may promote the healing of 5 wounds or the rate of recovery from infectious diseases such as pneumonia. Substances that inhibit the pro-inflammatory effects of chemokines ^, -, may be useful in treating pathological conditions mediated by inflammation such as arthritis, Crohn and other autoimmune patients. In addition, the established correlation between chemokine expression and inflammatory conditions and disease states provides diagnostic and prognostic indications for the use of chemokines, as well as as for antibody substances that are specifically immunoreactive with chemokines; There is a need for the identification and isolation of new chemokines to facilitate these diagnostic and prognostic indications. 20 Due to all the reasons mentioned above, there is a need for recombinant production methods of newly discovered chemokines, whose methods facilitate clinical applications involving chemokines and / or inhibitors of chemokines. chemokine.

* COMPENDIUM OF THE INVENTION The present invention fills one or more of the needs outlined above, providing purified and isolated polynucleotides encoding a human C-C chemokine designated Exodus, and fragments and ^ analogues thereof; purified and isolated Exodus polypeptides, fragments and analogs thereof; materials and methods for the recombinant production of these polypeptides, fragments and analogs thereof; antibodies to these Exodus polypeptides and the like, pharmaceutical compositions comprising these polypeptides, fragments, analogs or antibodies, and treatments using these polypeptides, fragments, analogs or antibodies, including prophylactic and therapeutic treatment. Exodus is a member of the C-C chemokine family that is expressed preferably in lymphocytes and monocytes, and is markedly upregulated by inflammatory stimuli. The deduced amino acid sequence of the cDNA encoding the Exodus is ninety-five amino acids longitude of which the first twenty-two N-terminal residues comprise a signal sequence. Their biological activities as demonstrated herein are to be expected to make it useful in a number of different clinical applications. Like other C-C chemokines, it stimulates the chemotaxis of mononuclear cells. Significantly, Exodus inhibits proliferation of the hematopoietic progenitor cell and also inhibits HIV production in infected cells. The invention specifically provides: purified polynucleotides (ie, DNA and RNA, both sense and antisense strands) encoding the Exodus amino acid sequence of SEQ ID NO: 2, particularly a DNA comprising a nucleotide sequence consisting of the coding portion of Exodus protein (nucleotides 43 to 327) of the nucleotide sequence of SEQ ID NO: 1; the purified polynucleotides encoding amino acids 1 to 73 of SEQ ID NO: 2, particularly a DNA comprising a nucleotide sequence consisting of nucleotides 109 to 327 of SEQ ID NO: 1; and purified polynucleotides that encode a full-length Exodus that is selected from the group consisting of: (a) nucleotides 43 to 327 of DNA of SEQ ID NO: 1; (b) a polynucleotide that hybridizes under stringent conditions in the complementary strand of nucleotides 43 to 327 of the DNA of SEQ ID NO: 1, or that would hybridize if the same under stringent conditions except as regards degeneracy of the genetic code; and (c) a polynucleotide encoding the same Exodus polypeptide as nucleotides 43 to 327 of DNA of SEQ TD N0: 1. The invention also provides vectors comprising these polynucleotides, particularly expression vectors wherein DNA encoding Exodus is operably linked to an expression control DNA sequence, host cells transformed or stably transfected with this DNA polynucleotide, and corresponding methods to produce Exodus by culturing these host cells and isolating the Exodus from host cells or their nutrient medium. The invention further provides purified Exodus polypeptides, particularly a polypeptide comprising the sequence Amino acid of SEQ ID NO: 2, or a polypeptide comprising amino acids 1 to 73 of SEQ ID NO: 2. Another aspect of the invention provides antibodies specifically reactive with Exodus, including monoclonal antibodies and hybridoma cell lines that produce these antibodies monoclonal. Yet a further aspect of the invention provides a method for increasing resistance to HIV infection by administering to a patient an amount of the Exodus protein product effective to inhibit proliferation of HIV, particularly when the patient is at risk of being exposed to HIV, or has been exposed to HIV, or has been infected with HIV. This aspect of the invention also provides a method of treating HIV infection which comprises administering to an HIV-infected patient an amount of the Exodus protein product effective to inhibit the proliferation of HIV. A further aspect of the invention provides a method for protecting the progenitor cells of the bone marrow from the cytotoxic effects which comprises administering an amount of the Exodus protein product effective to suppress the proliferation of the bone marrow progenitor cell, particularly when the patient is undergoing chemotherapy or radiotherapy. Yet a further aspect of the invention provides a method for treating myeloproliferative diseases comprising administering an amount of the Exodus protein product effective to suppress the proliferation of the malignant bone marrow progenitor cells. The invention is described more fully below. The invention provides purified and isolated polynucleotides (ie, DNA and RNA, both sense and antisense chains) encoding Exodus. Preferred DNA sequences of the invention include the genomic and cDNA sequences and the chemically synthesized DNA sequences.

The nucleotide sequence of an -DNAc encoding this Exodus chemokine, including 5 'and 3"non-coding sequences is set forth in SEQ ID NO: 1. Nucleotides 43 to 327 comprise the coding portion of the Exodus protein of this DNA of SEQ ID NO: 1, and a preferred DNA of the present invention comprises nucleotides 109 to 327 of SEQ ID NO: 1, which comprise the putative coding sequence of the mature secreted Exodus protein without its signal sequence. amino acid of the chemokine Exodus is set forth in SEQ ID NO: 2. Preferred polynucleotides of the present invention include, in addition to those polynucleotides described above, the polynucleotides encoding the amino acid sequence set forth in SEQ ID NO: 2, and that they differ from the polynucleotides described in the preceding paragraphs only because of the well-known degeneracy of the genetic code. idos (positions -22 to -1) of SEQ ID NO: 2 comprise a signal peptide that is cleaved to yield mature Exodus chemokine, preferred polynucleotides include those encoding amino acids 1 to 73 of SEQ ID NO: 2. this way, a preferred polynucleotide is a purified polynucleotide that encodes a polypeptide having an amino acid sequence comprising amino acids 1 to 73 of SEQ ID NO: 2. Among the uses for the polynucleotides of the present invention is the use of hybridization probes, to identify and isolate the genomic DNA encoding the Human exodus, whose gene has the possibility of having a structure of three exon / two intron characteristic of the ¿C-C chemokine genes (see from Baggiolin et al., Supra); in order to identify and isolate non-human genes that encode proteins homologous to Exodus; to identify the human and non-human chemokines that are similar to Exodus; and to identify those cells expressing Exodus and the conditions under which this protein is expressed. 15 Therefore, in another aspect, the invention "Provides a purified polynucleotide that hybridizes under stringent conditions in the complementary strand of the Exodus coding portion of the DNA of SEQ ID NO: 1. Similarly, the invention provides a purified polynucleotides which, except for the redundancy of the genetic code, would hybridize under stringent conditions in the complementary strand of the Exodus coding portion of the DNA of SEQ ID NO: 1. Hybridization conditions stringent specimens are the following: hybridization at 42 ° C in 5X SSC, 20 mM Nap04, pH 6.8, 50 percent formamide; and washed at 42 ° C in 0.2X SSC. Those skilled in the art will understand that it is desirable to vary these conditions based empirically on the length and base content of the GC nucleotide of the sequences to hybridize, and that there are formulas for determining this variation. [See, e.g. from Sambrook et al., Molecular Cloning: a Laboratoy Manual. Second Edition, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory (1989).] In another aspect, the invention includes vectors of Plasmid and viral DNA incorporating DNA of the invention, including any of the DNAs described above. Preferred vectors include expression vectors wherein the incorporated Exodus coding cDNA is operably linked to an endogenous or heterologous expression control sequence. These expression vectors may further include polypeptide encoding DNA sequences operably linked to the Exodus coding DNA sequences, which vectors may be expressed to yield a fusion protein that encodes the Exodus polypeptide of interest. In another aspect, the invention includes a prokaryotic or eukaryotic host cell transfected or stably transformed with a DNA or vector of the present invention. In preferred host cells, the Exodus polypeptide encoded by the DNA or vector of the invention is expressed. The DNAs, vectors and host cells of the present invention are useful, e.g., in methods for the recombinant production of large quantities of Exodus polypeptides of the present invention. These methods are themselves aspects of the invention. For example, the invention includes a method for producing Exodus wherein a host cell of the invention is grown in an appropriate nutrient medium and the Exodus protein. is isolated from the cell or the medium. In still another aspect, the invention includes the purified and isolated Exodus polypeptides. A preferred peptide is a purified chemokine polypeptide having an amino acid sequence comprising the amino acids from 1 to 73 of SEQ ID NO: 2. The polypeptides of the present invention can be purified from natural sources or provenances, but are preferably produced by recombinant methods using the DNA, vectors and / or host cells of the present invention , or synthesize chemically. The purified polypeptides of the invention can be glycosylated (e.g., O-linked or N-linked) or non-glycosylated, soluble or insoluble in water, oxidized or reduced, etc., depending on the host cell selected, the method of recombinant production, the method of isolation, processing, storage stabilizing agent, and the like. Alternatively, Exodus polypeptides can be prepared by synthesis of the chemical peptide using techniques that have been successfully used for the production of other chemokines, such as IL-8 [Clark-Lewis, et al., J. Biol. Chem., 266: 23128-34 (1991)] and MCP-1. ^ __- The invention also proposes Exodus polypeptide fragments, wherein one or more residues of N-terminal or C-terminal amino acids are deleted and retain one or more of the biological activities characteristic of C-C chemokines. Another aspect of the invention includes Exodus polypeptide analogs wherein one more amino acid residue is added, deleted or replaced from the Exodus of the present invention, and that retains one or more of the biological activities characteristic of the C-C chemicals. These analogs are useful for e.g., the medical imaging methods described above or the methods are treatment described below. They can be prepared by any of the recombinant or synthetic methods known in the art including those described below in Example 7. Exemplary analogs include substitutions in the Exodus amino acid sequence designed to effect greater homology with the chemokines with which it is more closely related. Substitutions designed to effect greater homology with the C-C chemokine family include replacing alanine at position 31 in the mature protein sequence with a threonine, or replacing phenylalanine at position 26 with a tyrosine. Other substitutions that would effect greater homology with MlP-la, MlP-lß and RANTES include replacing residues 1 to 8 of Exodus with residues 1 to 10 of MlP-la or residues 1 to 9 of RANTES, replacing leucine in position 11 with a phenylalanine, replacing glycine at position 12 with a serine, replacing glycine at position 25 with a glutamic acid, replacing glutamic acid at position 36 with a serine, replacing serine at position 46 with a glutamine, replacing isoleucine at position 60 with a tyrosine, and replacing serine at position 67 with an aspartic acid. These substitutions can be made individually or in combinations and are expected to have a potential to improve Exodus activity in myelosuppression or inhibition of HIV production. Other substitutions designed to improve the properties of an amino acid in a given position (eg, if an amino acid is hydrophobic, replenishment must be more hydrophobic) can also improve Exodus activities: replacing asparagine in position 6 with an aspartic acid , replacing leucine in position 18 with an isoleucine, replacing glutamine in position 29 with a glutamic acid, replacing asparagine in position 38 with aspartic acid, replacing valine in position 50 with isoleucine, and replacing glutamine in position 56 with glutamic acid. These substitutions can be made individually or in all combinations. A related aspect of the invention includes analogs lacking the biological activities of Exodus, but which are capable of competitively or non-competitively inhibiting the binding of C-C chemokines with their receiver (s). These analogs are useful, e.g., in therapeutic compositions or methods for inhibiting the biological activity of endogenous Exodus or other C-C chemokines in a host. These Exodus polypeptide analogs are specifically proposed to modulate the characteristics of binding Exodus to chemokine receptors and / or other molecules (eg, heparin, glycosaminoglycans, erythrocyte chemokine receptors) that are considered to be important in presenting Exodus to its receptor.

In related aspects, the invention provides purified and isolated polynucleotides encoding these Exodus polypeptide analogues, which polynucleotides are useful for, e.g. recombinantly producing the Exodus polypeptide analogs; plasmid and viral vectors incorporating these polynucleotides; and the prokaryotic and eukaryotic host cells stably transformed with these DNA or vectors. In another aspect, the invention includes antibody substances (e.g., monoclonal and polyclonal bodies, single chain antibodies, chimeric or humanized antibodies and the like) that are specifically immunoreactive with the Exodus polypeptides and polypeptide analogs of the invention. The invention further includes hybridoma cell lines that produce antibody substances of the invention. These antibodies are useful, for example, for purifying the polypeptides of the present invention for the detection or quantitative measurement of Exodus in fluid or tissue samples, e.g., using well-known ELISA techniques, and for modulating the binding from Exodus to its receiver (s). Some chemokine antibodies (e.g., anti-IL-8 antibodies) have been shown to have dramatic anti-inflammatory effects.

The recombinant Exodus polypeptides and polypeptide analogs of the invention can be used instead of the antibodies in binding reactions, to identify the cells expressing the Exodus receptor (s) and in normal expression cloning techniques to isolate the polynucleotides encoding the receptor (s). See, e.g. Example 16 presented below and the cloning of IL-8 and MCP-1 receptors in the article by Holmes et al., supra, and de Charo et al., supra, respectively. These Exodus polypeptides, Exodus polypeptide analogs and Exodus receptor polypeptides are useful for the modulation of the Exodus chemokine activity and for the identification of the polypeptide and chemistry (e.g., small molecule) of the Exodus agonist and antagonist. As used herein, "protein product Exodus "includes Exodus polypeptides, fragments or analogs thereof, alternatively including spliced Exodus variants, such as the chemokine Ckß-4 which is described in International Publication Number WO 96/05856, suspra, which retains the related biological activities of Exodus We have shown that the extra alanine found in Ckß-4 (after Exodus residue 4) remains within an intron-exon limit.The sequence through this region suggests that these two forms of Exodus are raised by alternative splicing.

The invention also proposes pharmaceutical compositions comprising Exodus protein products for use in methods for improving the immune response in a mammal suffering from a wound or an infectious disease. Pharmaceutical compositions comprising Exodus protein products or antibodies thereto are also proposed for use in methods to reduce inflammation or pathological conditions mediated by inflammation, such as arthritis, Crohn's disease, or other autoimmune diseases. Pharmaceutical compositions are also proposed to be used to reduce atherosclerosis, angiogenesis or growth or tumor metastasis. Pharmaceutical compositions are particularly proposed for use to suppress the proliferation of progenitor cells or hematopoietic hemocytoblasts. This myelosuppression can protect the stem cells or progenitor cells against the cytotoxic effects during chemotherapy or radiotherapy. The use of an Exodus protein product for the manufacture of a medicament for suppressing the proliferation of the bone marrow progenitor cell is also proposed, this drug being particularly desirable to be administered to a patient undergoing chemotherapy or radiotherapy.

- The pharmaceutical compositions for use in treating myeloproliferative diseases, and the use of an Exodus protein product for the manufacture of a medicament for treating myeloproliferative diseases are also particularly proposed. Pharmaceutical compositions are also specifically proposed for use in the treatment of patients recently exposed to HIV, but not yet tested or who have been confirmed as being HIV positive by normal diagnostic procedures, (e.g., neonates of HIV positive mothers, medical personnel exposed to HIV positive blood), patients at risk of HIV exposure, or patients already infected with HIV, ie, HIV positive patients. The use of an Exodus protein product for the manufacture of a medicament to inhibit the proliferation of HIV is also proposed, this drug being particularly desirable to be administered to patients who are at risk of being exposed to HIV, or exposed to HIV or infected with HIV. . The use of an Exodus protein product for the manufacture of a medicament for treating an HIV infection is also proposed. These pharmaceutical compositions comprise an Exodus protein product, or an antibody thereto with a physiologically acceptable diluent or carrier and may optionally include other appropriate therapeutic agents depending on the clinical application, e.g. anti-inflammatory agents or anti-HIV agents. The dosages of the Exodus protein product will vary between about 1 microgram to 100 milligrams per kilogram of body weight, preferably 5 to 100 micrograms per kilogram of body weight, depending , of the pathological condition that is going to be treated. These pharmaceutical compositions can be administered by a variety of routes depending on the condition to be treated including a subcutaneous, intramuscular, intravenous, intrapulmonary, transdermal, intrathecal, oral, or suppository administration. The dose of the Exodus protein product may be increased or decreased and the duration of treatment may be shortened or prolonged as determined by the physician treating the patient. The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the route of administration. The optimal pharmaceutical formulation will be determined by a person skilled in the art depending on the route of administration and the desired dosage. See for example, Remington's Pharmaceutical Sciences, Eighteenth Edition (1990, Mack Publishing Co., Easton, PA 18042) pages 1435 to 1712, the disclosure of which is incorporated herein by reference. These formulations can influence the physical state, the stability, the in vivo release regimen and the in vivo clearance regimen of the agents administered. Those skilled in the art will easily achieve effective dosages and simultaneous administration regimens as determined by good medical practice and the clinical condition of the individual patient. Independently of the In the manner of administration, the specific dose can be calculated according to the body weight, the area of the body surface or the size of the organ. Additional refinement of the calculations necessary to determine the appropriate dosage for the treatment which involves each of the formulations mentioned above, is routinely performed by those skilled in the art without undue experimentation especially in view of the dosing information and the assays disclosed herein, as well as the pharmacokinetic data observed in the human clinical trials discussed above. Appropriate dosages can be ensured through the use of established assays to determine dosages of blood levels along with the data of appropriate dose response. The final dosage regimen will be determined by the physician attending the case, taking into account several factors that modify the action of the drugs, e.g. the specific activity of the drug, the seriousness of the damage and the response of the patient, the age, the condition, the body weight, the sex and diet of the patient, the seriousness of any infection, the time of administration and other factors clinical Since studies are conducted, additional information is derived from appropriate dosage levels for the treatment of different diseases and conditions. The Exodus materials and methods described above can be used in various clinical applications. First, since chemokines attract and activate monocytes and macrophages (Baggiolini et al. above), the Exodus expression in a pathogenic inflammatory case may exacerbate the disease by recruiting additional monocytes and macrophages or other leukocytes to the site of the disease, activating the leukocytes that are already there, or inducing leukocytes to remain in place. the site. In this way, inhibiting the chemoraction activity of Exodus can be expected to alleviate the damaging inflammatory processes. Significantly, the potential benefits of this approach have been demonstrated directly in experiments involving IL-8, a chemokine C-X-C that attracts and activates neutrophils. Antibodies directed against 1L-8 have a profound ability to inhibit neutrophil-mediated inflammatory disease [Harada et al., J. Leukoc. Biol. 56: 559 (1994)]. Inhibition of Exodus is expected to have a similar effect in diseases where monocytes or macrophages are assumed to play a role, e.g. Crohn's disease, rheumatoid arthritis, atherosclerosis, myocardial infarction or acute respiratory distress syndrome (ARDS). 10 Alternatively, increasing the effect of Exodus may have a beneficial role in diseases, since chemokines have also been shown to have a positive effect on wound healing and angiogenesis. Therefore, protein products Exogenous Exodus or Exodus agonists can be beneficial to promote the recovery of these diseases. * ^^. The Exodus protein products or the Exodus agonists may also show to be clinically important in the treatment of tumors, as suggested by the The ability of the CC TCA3 chemokine to inhibit tumor formation in mice (see Laning et al., Supra), Exodus can act directly or indirectly to inhibit tumor formation, v.gr, by attracting and activating several effector cells not specific to the site of the tumor, or stimulating a specific tumor immunity. In addition, the deleterious effect shown here for Exodus indicates that the Exodus protein products or Exodus agonists can yield considerable benefits for patients receiving chemotherapy or radiation therapy, reducing the harmful effects of __ therapy in progenitor cells or I have acytoblasts in - myeloid of the patient. For example, treatment with Exodus protein product before or during (e.g., a day before, immediately before or at the same time) that the administration of chemotherapeutic agents specific to the cell cycle can protect the bone marrow against the cytotoxic effects of the agents.

These cell cycle-specific chemotherapeutic agents include vinblastine, etoposide, daunorubicin, doxorubicin, idarubicin, methotrexate, hydroxyurea, fluorouracil, cytosine arabinoside, mercaptopurine, thioguanine, pentostatin, fludarabine, and 2- 20 chlorodeoxyadenosine (2-CDA) . As discussed above, an analogue of MlP-la (designated BB10010, British BioTechnology) is currently in clinical trials as a myeloprotective agent in Cytoxan® (cyclophosphamide from Briston Myers Squibb Oncology) therapy.

- The ability of Exodus to inhibit the proliferation of cytokine-dependent myeloid cell lines as shown here, indicates that the Exodus protein product will also be useful in treating myeloproliferative diseases, including but not limited to chronic myelogenous leukemia, essential thrombocytosis. , myelofibrosis and polycythemia vera. The administration of the Exodus protein product for this object can be done simultaneously with the administration of other chemotherapeutic agents or other cytokines, such as interferon. In addition, the CC RANTES, MlP-la and MIP-Iß chemokines have been shown to suppress the duplication of the HIV-1 human immunodeficiency virus [Cocchi et al., Science, 270: 1811-1815 (1995)], implying the same as possible therapeutic agents in the prevention or treatment of AIDS. Exodus' ability to inhibit the proliferation of HIV, as demonstrated herein, indicates that the Exodus protein product will also be beneficial in treating AIDS patients, to prevent the initiation or progression of AIDS or to torment resistance to AIDS. HIV infection after exposure to HIV. Fully blown AIDS does not appear immediately during HIV infection, there being a variable period of time during which the patient remains healthy but exhibits viraemia. This viremia is sustained by continuous rounds of viral duplication and reinfection of blood cells. One study found that vital plasma face measurements (as well as CD4 lymphocyte counts) can predict the subsequent risk of AIDS or death. [Ho, Science, 272: 1124-1125 (1996).] Interference with the continuous cycle of viral duplication can therefore result in improved prognosis. In addition, the established correlation between chemokine expression and inflammatory conditions and disease states provides diagnosis and prognostic indications for the use of Exodus protein products, including antibody substances that are specifically immunoreactive with Exodus. These Exodus materials are useful in methods for diagnosing and valuing the prognosis of inflammatory conditions and disease states, as well as for medical imaging of areas involved in these conditions and disease states. Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art when taking into account the following detailed description of the invention which describes the currently preferred embodiments thereof.

# BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the effect of varying Exodus concentrations in mononuclear cell chemotax. Figure 2 shows the effect of untreated Exodus treated with ACN in hematopoiesis in mice. Figure 3 shows the effect of Exodus alone or with MCP-1 or MIG on the hematopoietic progenitor cell cycles in the mice. The figure 4 shows the effect of Exodus on the proliferation of the M07E myeloid cell line. Figure 5 shows the effect of Exodus on the proliferation of the myeloid TF-1 cell line. 15 Sample 6 shows the effect of purified synthetic Exodus on the proliferation of the M07E myeloid cell line. Figure 7 shows the effect of Exodus on the release of HIV p24 protein by mononuclear cells after HIV infection. Figure 8 shows the effect of purified synthetic Exodus protein product on the release of HIV p24 protein by mononuclear cells after infection with HIV. 25 # DETAILED DESCRIPTION OF THE INVENTION The invention is based on the identification of a cDNA sequence encoding Exodus and the characterization of Exodus activities. The complete cDNA of the Exodus chemokine is 821 nucleotides in length. There is a consensus polyadenylation site in 786. The sequence does not * translated 3 'has a sequence number AAAU that mediates the stability of mRNA in many cytokine genes. These sequences promote message degradation and contribute to the short half-life of many cytokine transcripts including chemokines. There is a region that does not move 5 'short of 43 nucleotides. 15 There are 95 amino acids in the Exodus sequence of deduced amino acids. This consists of the C-C chemokine family, where the length of the family members varies from 91 to 99. The first 22 amino acids of Exodus constitute an intensely signal peptide. hydrophobic. The four cysteines that participate in the disulfide bonds that define this family are also conserved in Exodus. Exodus is more closely related to MlP-la and RANTES at the amino acid level, with an identity of 26 percent to 28 percent, and approximately 75 percent similarity when # conservative changes are taken into account. The Exodus is especially similar to RANTES of amino acids 24 to 46 and again to 58 to 75, where between these positions there are only six non-conservative changes. While Exodus has many of the amino acid peculiarities conserved from the other human C-C chemokines, there are several unusual characteristics of Exodus that are worth noting. The Exodus * has a highly basic carboxy term, is more consistent with the MCP sub-family that RANTES. In addition, Exodus lacks a conserved tyrosine and a threonine at position 47 and 51, respectively, which are present in all other human C-C chemokines, including RANTES. It is not evident if these two conserved amino acids have a role in the activity of chemokine C-C, since it is not proclaimed to contact the recipient. Various aspects and advantages of the present invention will be understood by taking into account the following illustrative examples. Example 1 describes the identification of Exodus cDNA. Example 2 describes experiments that examine the pattern of Exodus gene expression in various lines of human cells and tissues. Example 3 describes the recombinant expression of the Exodus gene in mammalian cells. Example 4 describes another method for the recombinant expression of the Exodus gene in mammalian cells, and the purification of the resulting protein. Example 5 provides a protocol for the expression of the Exodus gene in the prokaryotic cells and the purification of the resulting protein. Example 6 provides a protocol for the recombinant production of Exodus in yeast or invertebrate cells. Example 7 describes the production of Exodus and Exodus analogs by peptide synthesis or recombinant production methods. He * Example 8 provides a protocol for generating antibodies monoclonal antibodies that are specifically immunoreactive with Exodus. Example 9 focuses on the effects of Exodus on monocyte chemotaxis in vitro. Example 10 focuses the Exodus effect on the proliferation of myeloid progenitor cells, myeloid cell lines and progenitor cells of chronic myelogenous leukemia. Example 11 sends the effect of Exodus on the production of p24 HIV protein. Examples 12, 13, 14 and 15 provide in vivo tests of the chemoattractant material of leukocyte activation, inhibition of tumor growth and activation of leukocyte after intraperitoneal or subcutaneous injection. Example 16 describes the cloning of an Exodus receptor.

EXAMPLE 1 Identification of the cDNA Sequence Encoding Exodus As described in the article by Takeda et al., Human Mol .. Genetics, 2: 1793-1798 (1993), the messenger 7? RN was prepared from the islet cells normal adult human pancreatic dissecans, and the first cDNA strand was synthesized by priming oligo (dT) using a primer containing an XhoI site. After the synthesis and mitigation of the second chain * CDNA, the EcoRI adapters were ligated to the cDNA which then was fractioned in size to remove products less than 1000 base pairs in size. After digestion of Xhol, the products were cloned into lambda ZAP II, and amplified in MRFT XLl-Blue cells, (Stratagene, La Jolla, CA). The library became plasmids rescuing pBluescript SK- according to the manufacturer's instructions. The partial sequences of 1000 of these randomly isolated pancreatic islet cDNAs were determined by automatic single pass sequence. These sequences were deposited in GenBank (Takeda et al. above) and compared with other sequences in the database of the National Center for Biotechnology Information (NCBI). The average length of the cDNA sequences used for comparison was approximately 200 bp. This work was pulped in Takeda and others, supra.

* Subsequently, to the work of Takeda and others, a clone encoding Exodus was identified among these 1000 Sequence Tag Epressed from pancreatic islet (EST) as follows. The comparison of a The sequence of consensual chemokine against these ESTs using the BLAST service of NCBl revealed that one of the clones possessed a distant histology to the chemokine family.

--- C-C. This homology of the chemokine family increased ~~~ r after several sequence errors from the step The original automatic sequence was identified by manual double-finger chain sequence, and the coding region and the reading frame were appropriately characterized.

This clone, originally designated HBC2850 by Takeda and others, was not identical to any of the other chemokine known. The cDNA of this clone consisted of 821 nucleotides containing the entire open reading frame of the chemokine protein. This chemokine is designated Exodus The differences between the Exodus cDNA sequence, which is set forth in SEQ ID NO: 1, and the EST sequence of Takeda and others, are as follows (with reference to nucleotide numbering according to SEQ ID NO: 1): in nucleotide 64 ("C" in Exodus), the nucleotide EST was "N"; in nucleotide 71 ("C" in Exodus), the nucleotide EST was 25"N"; between nucleotides 130 and 131, the EST contained a - 2 base "G" extra that caused a displacement in the reading box; between nucleotides 150 and 151, and the EST contained an extra "T" base that caused a shift in the reading frame; in nucleotide 193 ("C" in Exodus), nucleotide EST was "N"; in nucleotide 196 ("C" in Exodus), nucleotide EST was "N"; in nucleotide 271 ("A" in Exodus) the nucleotide EST was "N"; and at nucleotide 309 ("T" in Exodus), EST nucleotides # were "GC". EXAMPLE 2 Exodus Gene Expression Pattern in Cell and Tissue Lines The pattern of Exodus mRNA expression was examined through "Northern blotting" of the mRNA extracted from various human tissues and cell lines. The probe used was cDNA containing the complete coding region of Exodus isolated by gel electrophoresis agarose and irradiated with 32P-dCTP and 32P-dTTP (DuPont-NEN, Boston, MA) by random priming according to the manufacturer's instructions (BMB, Indianapolis, IN).

A. Exodus Gene Expression in Human Tissues RNA was isolated from cultured cell and monocyte lines using STAT-60 RNA (Tel-Test B Inc., Friendswood, TX) according to the manufacturer's instructions. Total RNA (20 micrograms) was fractionated into 0.5 0.8 percent agarose and formaldehyde gels, transferred to nitrocellulose, hybridized and washed under written conditions. The films were exposed for one day with an intensification screen at -80 ° C. A "Northern blot" of Human Multiple Tissue and a Multiple Tissue of the Northern Human Immune System (Clontech, Palo Alto, CA) were also tested with cDNA Exodus and were washed under strict conditions according to the manufacturer's instructions. The autoradiography was exposed as before for 1 to 4 days. 15 The Exodus seemed to have a very restricted pattern of expression. It was not expressed in a number of proven cell lines, including TMR323 neuroblastoma, MDA breast carcinoma, K562 erythroleukemia, Jurkat T cell leukemia, promyelocytic leukemia HL60, cells HL60 differentiated to granulocytes with retinoic acid, 3T3 embryonic fibroblasts or 293 embryonic kidney cells. When a commercially prepared "Northern blot" from a variety of normal human tissues was analyzed for Exodus expression, expression was detected in the lung and not in the heart, brain, placenta, adult liver, skeletal muscle, kidney, pancreas, spleen or bone marrow. The size of the transcript was approximately 0.9 kB, consistent with the size of the DNA, reported here given the addition of a poly A tail. However, when a commercially prepared "Northern blot" of the lymphoid tissues was examined for expression Exodus was found to be highly expressed in several different lymphoid organs. Exodus was highly expressed in peripheral lymph nodes, appendix, peripheral blood mononuclear cells and fetal liver. It was less highly expressed in the thymus, and there was no detectable expression in the spleen or marrow. This pattern of expression is typical of many chemokines. Exodus was expressed mainly in lymphoid tissue, especially in lymph nodes, the appendix, and peripheral blood. It is possible that the lymphoid tissue used in this "Northern blot" analysis may have been activated by a certain immunological stimulus, thus causing an expression higher than that of the expression level of Exodus Exodus deficient expression in the bone marrow as opposed that of peripheral blood was due to the fact that the bone marrow is composed mainly of immature myeloid precursors yerithroid, while there are much more mature mononuclear cells in the blood 1 peripheral.

B. Exodus Exodus Gene Expression After Inflammatory Stimulation Since the expression of many chemokines was induced in mononuclear cells by inflammatory stimuli, Exodus expression after exposure of several cell lines to LPS, TNF-alpha, or 10 PMA it was analyzed by "Northern blot" analysis. The THP-1 monocytic cell line was obtained from American Type Culture Collection (of Rockvielle, MD).

Cells were maintained in RPMI 1640 medium (Biowhitaker, Walkersville, MD) supplemented with 10 per percent fetal calf serum (FCS, Hyclone Laboratories, Inc., Logan, Uthah), 25 mM HEPES, 100 units per milliliter of penicillin and 100 micrograms per milliliter of streptomycin (tissue culture antibiotic, Life Technologies, Gaithersburg, MD). For stimulus experiments the cells were cultured at a density of one million cells per milliliter in the presence of forból ester (PMA, Sigma, St. Louis, MO). The endothelial cell line of the immortalized human umbilical vein I-HUVEC was obtained from Dr.

Jay Nelson, from the University of Oregon and was cultured in "• RPMI 1640 supplemented with 10 percent FCS (Hyclone), 400 micrograms per milliliter of G418 (Life Technologies, Grand Island, NY), one unit per milliliter of heparin (Sigma), and 30 micrograms per milliliter of endothelial cell growth factor (Collaborative Biomedical Products, Bedford, MA) to a confluence of 70 percent to 80 percent, and then cultured in the presence or absence of 10 ng per milliliter of tumor necrosis factor alpha (TNF-a, Peprotech, NJ) for several periods of time. Peripheral blood mononuclear cells were purified on Histopaque gradients (Sigma) and monocytes were isolated by plastic adhesion. The monocytes were cultured for six days with means that were replaced every two days to allow differentiation into macrophages. The cells were stimulated with 100 ng per ililliter of lipopolysaccharides (LPS, Sigma) for several periods of time. Exodus expression was highly induced when peripheral blood mononuclear cells were exposed to LPS for 8 or 12 hours. Exodus expression was again highly induced when the endothelial cells of the umbilical vein were exposed to TNF-a for only three hours. Significantly, Exodus expression remained elevated as long as inflammatory stimuli were present. When the THP-1 monocytic leukemia cell line was treated with PMA Exodus expression was also induced reached its maximum at 48 hours after exposure, and declining slightly later. These results indicated that Exodus was poorly expressed unless inflammatory stimuli were present. However, once a # stimulus, Exodus was rapidly ascending rapidly and stable. The nature of the stimulus itself also seemed to be not restricted with LPS, TNF-a and PMA all up-regulating Exodus. The production of Exodus therefore seems to be a function of mature lymphophagocytic cells, especially after inflammatory stimuli, and not myeloid and mature cells.

EXAMPLE 3 Production of Exodus Recoiribinant in COS Cells 20 The recombinant Exodus was produced transiently transfecting the cDNA in COS cells. The full length Exodus cDNA was subcloned using common restriction sites to the polylinker site of pECE [Ellis and others, Cell, 45: 721 (1986)] an expression vector driven by SV-40, in sense orientation. The logarithmic phase COS cells (American Type Culture Collection (ATCC) No. CRL 1651) were placed in DMEM with 10 percent FCS (Hyclone) and 100 units per milliliter of penicillin and 100 micrograms per milliliter of streptomycin (culture antibiotics) of tissue, Life Technologies, from Gaithersburg, MD) at a density of one million cells per 100 millimeter culture vessel and incubated overnight. Twenty micrograms of the plasmid DNA Exodus-pECE purified per plate was used for transfection of the COS cells with Lipofectin according to the manufacturer's instructions (Life Technologies, Bethesda, MD). The purified pECE plasmid (without the Exodus DNA was identically transfected in the COS cells to serve as a control.) An expression vector with the beta-galactosidase of the gene (SV40 / beta-Gal, Pharmacia, Piscataway, NJ) was co-transfected to control for transfection efficiencies Seventy-two hours later, the supernatant liquid from the COC cell culture was filtered through 0.2 micron filters and stored at -70 ° C. After separation of the supernatant liquid, the lysates from Cells were carried out and the beta-galactosidase activity was assayed as described above in Rosenthal, Meth. Enzymol., 152: 704 (1987) .When the transferences of pECE and pECE-Exodus were carried out, the efficiencies of transfection side by side as determined by beta-galactosidase activity, were within 10 percent of one with respect to the other.

EXAMPLE 4 Production of Exodus Recombinant in CHO Cells and Purification thereof. # 10 PCR was used to amplify bases 30 to 330 of the Exodus cDNA (shown in SEQ ID NO: 1), which includes 13 bp of the 5 'non-coding sequence and 3 bp of the 3' non-coding sequence. The sequences of the PCR primers were: 5 '-GGCGAAGCTTTGAGCTAAAAACCA G (SEQ ID NO: 3) and 5 '-GCGGGAATTCTTACATGTTCTTGACT (SEQ ID NO: 4). To facilitate cloning, these primers include the HindIII and EcoRI restriction sites, respectively (shown in italics). The fragment was cloned into the pDCl vector (described in the North American patent application co-pending co-owned Serial No. 08/558, 658 filed on November 16, 1995, incorporated herein by reference), which is a derivative of pBR322 that contains the immediate anterior CMV promoter adjacent to the cloning site to facilitate the expression of It also contains the bacterial beta-lactamase gene and the murine dihydrofolate reductase gene (DHFR) to allow selection of the plasmid in the bacterial and mammalian cells respectively (Sambrook et al., Supra). The construction containing the Exodus insert was aligned by restriction digestion with Pvul (BMB, Indianapolis, IN), which is segmented within the vector sequence. The aligned plasmid was precipitated with ethanol and redissolved in HBS (20 mM HEPES-NaOH, pH 7.0, 137 mM NaCl, 5 mM KCl, 0.7 mM Na HP0, 6 mM Dextrose). For electroporation, 10 ^ cells of the CHO DG44 cell line [Urlab et al., Cell, 33: 405 (1983)], were washed, resuspended in one milliliter of PBS, mixed with 10 micrograms of the aligned plasmid, and transferred to a 0.4 cm electroporation cuvette. The suspension was electroporated with a Biorad Gen Button (Richmond, CA) at 290 volts, 960 microFarads. Transformants were selected by growth in the DMEM / F12 medium (Gibco) containing 10 percent dialyzed FC (Hyclone, Logan, UT) and lacking hypoxanthine and thymidine. Cells from several hundred transformed colonies were pooled and re-treated in the DMEM / F12 medium containing 20 nM methotrexate (Sigma, St. Louis, MO). Colonies surviving this round selection were isolated and expanded to obtain individual clones. The level of Exodus expression was determined as follows. The clones were grown in tissue culture plates to approximately 90_ percent confluence in the DMEM / F12 medium containing 10 percent dialyzed FCS, at which time the medium was replaced. The cells were allowed to grow for 4 days in the DMEM / F12 medium containing 1 percent dialyzed FCS. The supernatant was loaded onto a Heparin Sepharose CL-6B column (Pharmacia, Piscataway, NJ). The column was raised with 0.2 M NaCl in 20 M Tris, pH 7.5, and the chemokine was eluted with 0.6 M NaCl in 20 mM Tris, pH 7.5. The eluted Exodus was fractionated by SDS-PAGE through an 18 percent Tris glycine gel (NOVEX, San Diego, CA) and transferred to a PVDF membrane (Millipore, Bedford, MA). The Exodus band migrating at approximately 7 kD was confirmed by detection with the Exodus-specific rabbit polyclonal antiserum (prepared as described in Example 8 below). Clones that express the highest level of the Exodus chemokine can be expanded for large-scale protein production. The resulting recombinant Exodus was produced and purified from the supernatant liquid in the following manner. The Exodus band migrating at approximately 7 kD is cut off and the N-terminus is sequenced in an automatic sequence apparatus (Applied Biosystems, Model 473A, Foster City, CA). As an additional purification step, the Exodus eluted from the Heparin-Sepharose column is brought up to 1.6 M NaCJ and loaded onto a 40 micron HI-Propyl resin column (J.T. Baker, Phllipsburg, NJ). The column is washed with 1.6 M NaCl in 20 mM Tris, pH # 7.5 and the Exodus is eluted with 20 mM Tris, pH 7.5. 10 The integrity of the eluted Exodus is verified by amino acid analysis to confirm the ratio of the amino acids predicted by the protein sequence by mass spectrometry to confirm the predicted size. fifteen Example 5 Production of Recombinant Exodus in Bacteria Exemplary protocols for recombinant expression of Exodus in bacteria and purification of the resulting product are given below. The DNA sequence that encodes the mature form of the protein is amplified by PCR and cloned towards the pGEX-3X vector (Pharmacia, Piscataway, NF). The pGEX vector is designed to produce a fusion protein comprising glutathione-S-transferase (GST), encoded by the vector and a protein encoded by the DNA fragment inserted at the cloning site of the vector. The primers for PCR are SEQ ID NO: 4 and 5 '-TAT CGG ATC CTG GTT CCG CGT GAA TCA GAA GCA AGC AAC T-3 *, which includes a restriction site BamHl, a thrombin cleavage site [Chang, Eur J. Biochem., 151: 217 (1985)], and nucleotide 109 to 127 of SEQ ID NO: l. The resulting PCR product is digested with BamHl and EcoRI and inserted into a pGEX-3X plasmid with BglII and EcoRI. The treatment of the recombinant fusion protein with thrombin or factor Xa (Pharmacia, Piscatawayt, NJ) is expected to segment the fusion protein, releasing the chemokine from the GST portion. The construction of pGEX-3X / Exodus is transformed into E. coli XL-1 Blue cells (Stratagene, La Jolla CA), and the individual transformants were isolated and grown. The DNA plasmid of the individual transformants is purified and partially sequenced using an automatic sequence apparatus to confirm the presence of the desired Exodus gene insertion in the proper orientation. Induction of GST / Exodus fusion protein is achieved by growing the XL-1 Blue transformer culture at 37 ° C in the LB medium (supplemented with carbenicillin) to an optical density at a wavelength of 600 nm of 0.4, followed by additional incubation for 4 hours in the presence of 0.5 mM of 5-Dipropyl β-D-thiogalactopyranoside (Sigma Chemical Co., St. Louis MO). The fusion protein, expected to be produced as an inclusion body insoluble in the bacteria, can be purified in the following manner. The cells are - '' harvested by centrifugation, washed in 0.15 M NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA; and treated with 0.1 milligram per milliliter of lysozyme (Sigma Chemical Co.) for 15 minutes at room temperature. The lysate is cleared by sonification and the cell waste is granulated by centrifugation for 10 minutes at 12,000 X g. The granule containing the fusion protein is resuspended in 50 mM Tris, pH 8, and 10 mM EDTA, layered through 50 percent glycerol, and centrifuged for 30 minutes at 6000 X g. The granule is resuspended in a saline solution stabilized with phosphate normal (PBS) free of Mg ++ and Ca ++. The fusion protein is further purified by fractionation of the resuspensed granule in a denaturing SDS polyacrylamide gel (Sambrook et al., Supra). The gel is soaked in 0.4 M KCl to visualize the protein that is short and electroeluted in a gel stabilizer lacking SDS. If the GST protein / Exodus fusion bacteria are produced as a soluble protein, it can be purified using a GST Purification Module (Pharmacia Biotech). The fusion protein can be digested of thrombin for GST segmentation of the mature Exodus protein. The digestion reaction (from 20 to 40 micrograms of fusion protein, from 20 to 30 units of human thrombin (4000 units per milligram (Sigma) in 0.5 milliliter of ^ F PBS) is incubated for 16 to 48 hours at room temperature environment and loaded on a denaturing SDS-PAGE gel to fractionate the reaction products. The gel is soaked in 0.4 M KCl to visualize the protein bands. The identity of the protein band corresponding to the expected molecular weight of Exodus can be confirmed by partial analysis of ^. amino acid sequence using an automatic sequence apparatus (Applied Biosystems Model 473A, Foster City, CA). Alternatively, the DNA sequence that encodes the predicted mature Exodus protein can be cloned into a plasmid containing a desired promoter and, optionally, a forward sequence [see, e.g., Better et al., Science, 240: 1041-423 (1988)]. The sequence of this construction can be confirmed by a automatic sequence. The plasmid is then transformed into strain E. coli MC 1061 using normal procedures employing CaCl 2 incubation and heat shock treatment of the bacteria (Sambrook et al., Supra). The transformed bacteria are grown in the LB medium supplemented with carbenicillin, and the production of the expressed protein is induced by growth in an appropriate medium. If present, the forward sequence will effect the secretion of the mature Exodus protein and will be segmented during secretion. The secreted recombinant protein is purified from the bacterial culture medium by the method described above in Example 4 or, e.g., by adapting methods described above for the purification of recombinantly produced RANTES chemokine [Kuna et al. , J. Immunol. , 1 49: 636-642 (1992)], MGSA chemokine [Horuk et al., J. Bi ol. Chem. 268-541-46 (1993)], and IP-10 chemokine (expressed in insect cells) [Sarris et al., J. Exp. Med., 178: 1127-1132 (1993)].

EXAMPLE 6 Recombinant Exodus Production in Invertebrate Cells or Yeast Exemplary protocols for the recombinant expression of Exodus in yeast or invertebrate cells and for the purification of the resulting recombinant protein are given below. The Exodus cDNA coding region is amplified by PCR. A DNA encoding the pre-pro-alpha yeast forward sequence is amplified from the yeast genomic DNA in a PCR reaction using a primer containing nucleotides 1-20 of the alpha mating factor gene and another primer complementary to the ¥ nucleotides 255-235 of this gene [Kurkan and Herskowitz, Cell, 30: 933-943 (1982)]. The pre-pro-alpha forward coding sequence and the Exodus coding sequence fragments are ligated into a plasmid containing the yeast alcohol dehydrogenase (ADH2) promoter, such that the promoter directs the expression of a A fusion protein consisting of the pre-pro-alpha fused factor in the mature Exodus polypeptide. As disclosed by Rose and Broach, Meth. Enz. 185: 234-279, D. Goeddel, editor, Academic Press, Inc., San Diego, CA (1990), the vector also includes a terminator ADH2 transcription downstream of the cloning site, the duplication origin of the "2-micron" yeast, the yeast gene leu-2d, the yeast REP1 and REP2 genes, the E. coli beta-lactamase gene and an origin of E. coli duplication. The beta-lactamase and leu-2d 25 genes provide selection in bacteria and yeasts, respectively. The leu-2d gene also facilitates the increased copy number of the plasmid in yeast to induce higher levels of expression. The REP1 and REP2 genes encode the proteins involved in the regulation of the copy number of the plasmid. The DNA construct described in the previous paragraph is transformed into yeast cells using a known method e.g., treatment with lithium acetate [Stearns et al. , Meth. Enz. , supra, pages 280 to 297]. The ADH2 promoter is induced during glucose depletion in the growth medium [Price et al., Gene, 55: 281 (1987)]. The pre-pro-alpha sequence affects the secretion of the fusion protein of the cells. Concomitantly, the KEX2 yeast protein segments the pre-pro sequence of the mature Exodus chemokine [Bitter et al., Proc. Nati Acad. Sci. USA, 81: 53330-5334 (1984)]. Alternatively, Exodus is recombinantly expressed in yeast using a commercially available expression system, e.g., the Pichia Expression System (Invitrogen, San Diego, CA), following the manufacturer's instructions. This system also depends on the pre-pro-alpha sequence to direct the secretion but the transcription of the insert is driven by the alcohol oxidase promoter (AOX1) during the induction by methanol.

The secreted recombinant Exodus is purified from the yeast growth medium by, eg, those used to purify the Exodus from the supernatants of the bacterial and mammalian cells (see Examples 4 and 5 above). Alternatively, the cDNA encoding Exodus is cloned into the baculovirus expression vector ?? pVL1393 (PharMingen, San Diego, CA). This vector which contains Exodus is then used according to the manufacturer's instructions (PharMingen) to infect the Spodoptera frugiperda cells in the sF9 protein-free media and to produce recombinant protein. The protein is purified and concentrated from the medium using a heparin-Sepharose column (Pharmacia, Piscataway, NJ) and sequential molecular size columns (Amicon, Beverly, MA), and resuspended in PBS. The SDS-PAGE analysis shows a single band and confirms the size of the protein, and the EDman sequence in a 2090 Peptide Gate Sequence apparatus confirms its terminal N-20 sequence.

EXAMPLE 7 Production of Exodus Analogs Recombinant techniques such as those described in the previous examples can be used to prepare analogs of the Exodus polypeptide. More particularly, the polynucleotides encoding the Exodus are modified to encode analogs of the polypeptide of interest using well known techniques, e.g., site-directed mutagenesis and polymerase chain reaction. See generally Sambrook et al., Supra, Chapter 15. The modified polynucleotides are expressed recombinantly, and the recombinant polypeptide analogs are purified as described in the previous examples. Critical residues for Exodus activity are identified, eg, by homology to other C-C chemokines and by suspending alanines by the residues of the native Exodus amino acid. Cysteines are often critical to the functional integrity of proteins due to their ability to form disulfide bonds. To determine if any of the four cysteines in Exodus is critical for enzyme activity, each cysteine undergoes mutation individually to a serine. Other exemplary analogs include substitutions in the Exodus amino acid sequence designed to effect greater homology with the chemokines to which it is more closely related. Substitutions # designated to effect greater homology with the C-C chemokine family include replacing alanine at position 31 in the mature protein sequence with a threonine, or replacing phenylalanine at position 26 with a tyrosine. Other substitutions that would effect greater homology with MlP-la, MlP-lß and RANTES include replacing residues 1-8 of Exodus with residues 1-10 of MlP-la or residues of 1-8 of RANTES, replacing the ^^ leucine in position 11, with a phenylalanine, replacing glycine in position 12 with a serine, replacing glycine in position 25 with a glutamic acid, replacing glutamic acid in position 36 with a serine, replacing serine in position 46 with glutamine, replacing isoleucine in position 60 with a tyrosine, and replacing the serine at position 67 with an aspartic acid. These substitutions can be made individually or in all combinations and are expected to have a potential to improve Exodus activity in myelosuppression or inhibition of production of HIV. Other substitutions designed to improve the properties of an amino acid in a given position (eg, if an amino acid is hydrophobic, the replenishment is going to be more hydrophobic) they can also improve the activities of Exodus: replacing asparagine in position 6 with an aspartic acid, replacing leucine in position 18 with an isoleucine, replacing glutamine in position 29 with a glutamic acid, replacing asparagine in position 38 with aspartic acid, replacing valine at position 50 with isoleucine, and replacing glutamine at position 56 with glutamic acid. These substitutions can be made individually or in all combinations. C-terminal deletions are prepared, e.g., By digesting the 3 'end of the Exodus aqe encodes the exonuciease III sequence for various time intervals and then by ligating the shortened coding sequence with the plasmid DNA encoding the stop codons in all three reading frames. HE prepare the N-terminal deletions in a similar manner by digesting the 5 'end of the coding sequence and then ligating the digested fragments into a plasmid containing a promoter sequence and initiating the methionine immediately upstream of the developer site. These analogs of N-terminal deletion can also be expressed as fusion proteins. Alternatively, Exodus polypeptide analogs can also be prepared by synthesis of the chemical peptide using techniques that have been used successfully for the production of other chemokines such as IL-8 [Clark-Lewis et al., J. Biol Chem., 266: 23128-34 (1991)] and MCP-1. These methods are advantageous because they are fast, reliable for short sequences such as chemokines, and allow the selective introduction of novel non-natural amino acids and other chemical modifications. The properties of Exodus analogues in one or more cell types involved in the process inflammatory cells (eg, T-lymphocytes, monocytes, macrophages, basophils, eosinophils, neutrophils, mast cells, endothelial cells, epithelial cells and others), are assayed by recognized techniques in the field that have been used to test these properties of numerous others chemokines. The properties of Exodus analogues to inhibit myeloproliferation and HIV production also * are tested according to Examples 10 and 11 which are presented below.

EXAMPLE 8 Preparation of Antibodies to Exodus Exodus chemokine was synthesized chemically in an essential manner as described in Example 7. For storage, Exodus was diluted in the RPMI medium which - It contains 1 percent bovine serum albumin (Sigma, St. Louis, MO). The Exodus was subsequently purified from the medium by passing through a Heparin Sepharose CL-6B column (Pharmacia, Piscataway, NJ). The column was washed with a solution of 0.2 M NaCl and 20 mM Tris, pH 7.5, and the chemokine was eluted with 0.6 M NaCl and 20 mM Tris, pH 7.5. To generate the polyclonal antisera, 50 micrograms of Exodus were emulsified with the Freund Complete Adjuvant for immunization of rabbits. At 21-day intervals, 50 micrograms of Exodus was emulsified in Freund's Incomplete Adjuvant for reinforcement. These antisera recognized the chemically synthesized Exodus and an Exodus derived from the CHO cell (prepared as described in Example 4) in Western blot. To generate monoclonal antibodies to Exodus, a mouse is periodically injected with recombinant Exodus (e.g., 10 to 20 micrograms emulsified in Freund's Complete Adjuvant) obtained as described in any of Examples 3 to 7. mouse a final Exodus pre-fusion booster in PBS, and four days later the mouse is sacrificed and its spleen is removed. The spleen is placed in 10 milliliters of serum-free RPMI 1640 and a single cell suspension is formed by grinding the spleen between the frozen ends of two glass microscope slides immersed in RPMI 1640 serum-free, supplemented with 2 mM glutamine , one mM of sodium pyruvate, 100 units per milliliter of penicillin and 100 micrograms per milliliter of streptomycin (RPMI) (Gibco, Canada). The cell suspension is filtered through the sterile 70 mesh Nitex cell strainer (Becton Dickinson, Parsippnay, New Jersey), and washed twice by centrifuging at 200 grams for 5 minutes and resuspending the pellet to 20 milliliters of RPMI. free of serum. Splenocytes taken from three Balb / c mice were prepared in a similar manner and used as a control. NS-1 myeloma cells maintained in the logarithmic phase in RPMI with 11 percent-fetal bovine serum (FBS) (Hyclone Laboratories, Inc., of Logan, Utah) for three days before melting, centrifuged at 200 grams for 5 minutes, and the pellet is washed twice as described in the previous paragraph. One per 10 ^ spleen cells are combined with 2.0 x 10 ^ NS ~ 1 cells and centrifuged and the liquid supernatant is aspirated. The cell pellet is dislodged by beating the tube and 1 milliliter of PEG 1500 at 37 ° C (50 percent in 75 mM Hepes, pH 8.0) is added with agitation (Boehringer Mannheim) through the course of 1 minute, followed by the addition of 7 milliliters of RPMI free of serum through 7 minutes. 8 milliliters of additional RPMI are added and the cells are centrifuged at 200 grams for 10 minutes. After discarding the supernatant fluid, the granule is resuspended in 200 milliliters of RPMI containing 15 percent FBS, 100 micrometers of sodium hypoxanthine, 0.4 micrometer of aminopterin, 16 micrometers of thymidine (HAT) (Gibco), 25 units per milliliter of IL-6 (Boehringer "Mannheim) and 1.5 x 106 splenocytes per milliliter and placed in 10 plates ^ Corning 96-well tissue culture with flat bottom (Corning, Corning New York). On days 2, 4 and 6, after the fusion, 100 microliters of the medium are removed from the wells of the fusion plates and replaced with a fresh medium. On day 8, the fusion is subjected to ELISA, testing for Determine the presence of the IgG mouse that binds to the Exodus in the following manner. Immulon 4 plates (Dynatech, ^ Cambridge, MA) are coated for 2 hours at 37 ° C with 100 ng per well of Exodus diluted in 25 mM Tris, pH 7.5. The coating solution is aspirated and 200 microliters per well of the blocking solution [0.5 percent fish skin gelatin (Sigma) diluted in CMF-PBS] is added and incubated for 30 minutes at 37 ° C. The plates are washed three times with PBS with 0.05 percent Tween 20 (PBST) and 50 microliters of the liquid are added. culture supernatant. After incubation at 37 ° C for 30 minutes, and washing as above, 50 microliters of horseradish peroxidase conjugated with goat anti-mouse IgG (fc) (Jackson ImmunoResearch, West Grove, Pennsylvania) diluted to 1 is added. : 3500 in PBST. The plates are incubated as above, washed four times with PBST and 100 microliters of the substrate consisting of 1 milligram per milliliter of o-phenylene diamine (Sigma) and 0.1 microliter per milliliter of 30 percent H2O2 in 100 mM Citrate , pH 4.5, are then added. The reaction Color 10 stops after 5 minutes with the addition of 50 microliters of 15 percent H2SO4. 490 is read on the plate reading device (Dynatech). The selected fusion wells are cloned twice by dilution in 96-well plates and the total visual of the number of colonies / well after 5 days. The monoclonal antibodies produced by hybridomas are Isotyped using the Isostrip system (Boehringer Mannheim, Indianpolis, IN).

Example 9 Effect of Exodus on Quimiotaxa from Monocyte.

The activity of Exodus was evaluated in chemotaxis assays carried out as described above in the article by Martinet et al., J. Immonol. Meth. , 1 74: 209, 1994 and Keller et al., J. Immunol. Meth. , 1: 165, 1972. Twenty milliliters of peripheral blood were collected from healthy volunteers in 10-milliliter heparinized tubes. The blood was diluted 1: 1 with PBS and then with 10 milliliters of Histopaque (Sigma). After centrifugation at 400 grams for 25 minutes, the cells at the interface were harvested and washed twice in PBS. The cells were resuspended in DMEM (Life Technologies, 9- Gaithersburg, MD) with 100 units per milliliters of penicillin and 100 micrograms per milliliter of streptomycin (tissue culture antibiotics, Life Technologies) at 10 ^ / milliliter. Sterile bovine serum albumin (Sigma) was added at a final concentration of 0.2 milligrams per milliliter. 15 100 microliters of this cell suspension was added to each transpozo insert (Costar). The DMEM with antibiotics and 0.2 percent BSA with or without pure synthetic Exodus was added to the lower wells in the 24-well plate. All concentrations of Exodus were carried out in triplicate. The transpozo insertions were placed in the lower walls and incubated at 37 ° C for 90 minutes at the end of the incubation period the inserts were removed and the upper part of the filter was scraped with a surgical device. rubber to remove adherent cells. The entire insert was then stained with Wright-Giemsa. Cells adhering to the lower surface of the insertion and those that migrated to the lower well were counted under high power 3 fields and added together to obtain a total number of migrating cells. The purified synthetic Exodus was tested at concentrations of 5, 50 and 500 ng per milliliter. For comparison, MlP-la was tested at a concentration of 833 ng per milliliter. The control did not contain chemokine.

The results are shown in Figure 1. The values represent the average of two experiments carried out in triplicate, plus or minus the normal error. The stars represent statistically significant differences? and control to p < 0.05 using the Student's t test.

These results show that Exodus stimulated the chemotactic activity of mononuclear cells of «R normal human peripheral blood as measured by transpozo migration. Higher concentrations of Exodus stimulated chemotaxis more efficiently than the maximum effective concentrations of MlP-la. Similar results were obtained with an Exodus protein product, which was Exodus with additional alanine after residue 4.

EXAMPLE 10 Effect of Exodus on Proliferation of Myeloid Cells A. Effect on Myeloid Progenitor Cells The effect of the Exodus protein products on the formation of the hematopoietic colony was tested essentially as described above, in e.g., Broxmeyer et al., Blood, 76: 1110 (1990). Bone marrow cells were collected from human donors after J- and to obtain informed consent. The cells of low-density human bone marrow at 5 x 10 ^ per milliliter were placed in 1 percent methylcellulose in the Iscove Modified Essential Medium (Biowhitaker, Walkersville, MD) supplemented with 30 percent FCS (Hyclone), human erythropoietin recombinant (EPO, 1 unit per milliliter, -Amgen of Thousand Oaks, CA), recombinant human interleukin-3 (IL-3, 100 units per milliliter, Immunex, Seattle, WA), and recombinant human hemocytoblast factor (SCF, 50 ng per milliliter , -Amgen) for granulocyte / colony forming unit macrophage (CFU-GM), granulocyte / erythrocyte / macrophage / megakaryocyte colony forming unit (CFU-GEMM) or erythrocyte unit formation analysis (BFU-E). The cultures were incubated at 5 percent C02 and low oxygen tension (5 percent) for 14 days, and then added to the formation of the colony using an inverted microscope in a blind manner. The experiments were carried out at least twice in triplicate. Variable amounts of supernatant liquid of the COS cells containing Exodus, prepared as described in Example 3 above, were tested in this assay, as was MlP-la (R & D Systems, Minneapolis, MN) at 50 ng per milliliter. The results are shown in Table 1 below, which presents the mean count of the hematopoietic progenitor colonies. per plate, plus or minus the normal deviation.

TABLE 1 Chemokine in the CFU-GM Medium BFU-E CFU-GEMM Control (without chemokine) 53 + 56 + 24 + 5 MlP-la 50 ng per milliliter 23 + 4 ^ 37 + 3 ^ 12 + 1 ' Exodus COS cell supernatant (0.2 milliliter in 2 milliliters of total medium) 21 + 3 * 29 + 2 * 11 + 1 ' # Exodus COS cell supernatant (0.1 milliliter in 2 milliliters 5 of the total medium) 24 + 4 * 27 + 5 * 11 + 2 * Exodus COS cell supernatant (0.05 milliliter in 2 milliliters 10 of the total medium) 23 + 5 * 36 + 3 * 14 + 1 * Exodus COS cell supernatant (0.025 milliliter in 2 milliliters 15 of the total medium) 47 + 8 58 + 6 24 + 1 Supernatant liquid of the COS cell only pECE (0.2 milliliter in 2 20 milliliters of the total medium) 50 + 6 60 + 7 23 + 3 * p < 0.005 (the other values are not significantly different from the control or from pECE at p <0.05) The Exodus in the supernatant of the COS cell inhibited the formation of the hematopoietic progenitor colony in a dose-dependent manner, slightly more efficiently of a maximum dose of MlP-la. There is no statistical difference between the COS cell medium alone from the medium of the COC cells that has been transfected with the empty pECE expression vector. At 50 ng per milliliter of the recombinant MlP-la, a dose at which the biological effect reaches a high plateau, statistically significant reduction of both CFU-GM (43 percent control of the medium), BFU-E (66 percent control), and CFU-GEMM (50 percent control). At the highest concentrations of recombinant Exodus used in these experiments there was also a decrease statistically significant in both CFU-GM (42 percent control), BFU-E (48 percent control), and CFU-GEMM (48 percent control). This inhibition by Exodus depended on the dose, since the three highest levels of Exodus showed inhibition of hematopoietic progenitor proliferation as measured by colony formation assays. However, the lowest concentration of Exodus used did not show this inhibition. Like MlP-la, Exodus inhibited progenitors in a way that multiple lineage.

# The purified synthetic Exodus was also tested in this test. The results are shown in Table 2 below, which presents the mean count of hematopoietic progenitor colonies per plate, plus or less the normal deviation.

TABLE 2 - Concentration of 10 Chemokine in CFU-GM Medium BFU-E CFU-GEMM Control (without chemokine) 85 + 3 97 + 4 39 + 3 Exodus (200 ng / milliliter) 43 + 11 43 + 2 19 + 3 Exodus (100 ng / milliliter) 39 + 3 41 + 2 20 + 2 Exodus (50 ng / milliliter) 42 + 10 42 + 3 17 + 2 Exodus (25 ng / milliliter) 41 + 2 50 + 2 20 + 4 Exodus (12.5 ng / milliliter) 51 + 3 70 + 10 27 + 1 Exodus (6.25 ng / milliliter) 64 +! 7 + 3 32 + 2 MIP-la (100 ng / milliliter) 41 + 2 44 + 3 19 + 2 IL-8 (100 ng / milliliter] 42 + 2 44 + 2 19 + 2 PF-4 (100 ng / milliliter) 42 + 4 44 + 5 19 + 1 RANTES (100 ng / milliliter) 81 + 7 99 + 4 37 + 1 NAP-2 (100 ng / milliliter) 83 + 2 93 + 3 39 + 4 There was a statistically significant reduction (Student's T test) in the colony formation of all three types at Exodus concentrations at 25 ng per milliliter (p <0.005). The purified synthetic Exodus behaves identically to Exodus in the supernatants of COS cells. Both sources of Exodus are effective to inhibit hematopoietic stem cell proliferation, at least also if not better than MlP-la. Similar results showing inhibition of proliferation of hematopoietic progenitors, as measured by colony formation assays, were obtained with a purified synthetic Exodus protein product, which were Exodus with additional alanine after residue 4. These results show that the Exodus protein products inhibited the proliferation of hematopoietic progenitors. Of course, the Exodus protein products were as effective as MlP-la. This indicates that the Exodus protein products will be useful as cycle-specific chemoprotective agents. f ^ "Additional experiments confirmed that the The effect of the Exodus protein product in vivo on hematopoiesis in mice. The experiments were carried out essentially as described in the article by Broxmeyer et al., Ann. Hematol. , 71: 235-246 (1995), using untreated pure synthetic Exodus and Exodus treated with a solution of 30 percent acetonitrile / 1 percent trifluoroacetic acid (ACN) as described in the article by Mantel et al., Proc. Na ti. Acad. Sci, USA, 90: 2232-2236 (1993). For chemokines that exist in solution as multimers, treatment with ACN stimulates the formation of monomers, which are the active form in vivo, and therefore improves the activity of chemokine. Chemokines treated with ACN can be active at concentrations 200 times lower than untreated chemokines.

Abbreviating, the solutions of the untreated Exodus or Exodus treated with ACN were prepared at concentrations ranging from 0.001 to 50 ng per milliliter of Exodus. The diluent treated with ACN or not treated served as a control to show that the ACN was not toxic. The normal C3H / HeJ mice were injected intravenously with a dose of 0.2 milliliter of each solution and sacrificed after 24 hours. Marrow cells were obtained * Bone not separated from your femurs and placed either in agar with 10 volume percent / volume of a pokeweed mitogen mouse spleen cell conditioned medium (PWMSCM), for CFU-GM titration, or in methylcellulose with human erythropoietin (Epogen®, Amgen, Thousand Oaks, CA ), PWMMSCM and hemina (Eastman Kodak Co., Rochester, NY), for valuation of BFU-E / CFU-GEMM. The colony counts were determined after seven days * of incubation in a humid environment in a ESPEC N2-02-C02 BNP-210 incubator (Taboi ESPEC Corp., South Plainfield, NJ) at 5 percent CO 2 and 5 percent O2. The results are presented in Figure 2. The results showed that the untreated Exodus reduced CFU-GM colony formation to an average of 56 percent of the control when a single 0.2 milliliter dose of 25 ng per milliliter was injected. Exodus He Exodus treated with ACN reduced the formation of the colony - í CFU-GM up to an average of 53 percent of control when a single 0.2 milliliter dose of 0.1 ng per milliliter of Exodus was injected. Inhibition of in vivo formation of CFU-GM induced by both Exodus treated with ACN and untreated was statistically significant at p < 0.05. In another experiment, the effect of untreated Exodus on the in vivo cycling of progenitors -f hematopoietic agents were evaluated with an extermination test Tritiated thymidine essentially as described in the Broxmeyer et al. Article, Ann. Hematol. , supra and in the article by Mantel et al., Proc. Na ti. Acad. Sci. , supra. Abbreviating the mice were treated with varying concentrations of untreated Exodus (0.5, 1 or 10 ng / milliliter) alone or in combination with other untreated chemokines (Exodus at 0.01 or 0.1 ng per milliliter with either MCP-1 or MIG at 0.01 or 0.1 ng / milliliter. The animals were then sacrificed after 24 hours and the bone marrow was collected for a tritiated thymidine killing assay.

Because the cells in the S phase of the cell cycle are preferentially incorporated thymidine, they are killed by the incorporation of tritiated thymidine while the cells that are not in the S phase do not experience any damage. The number of celias in the S phase is calculated calculating the number of killed cells, based on the control colony numbers of cells that were not treated with tritiated thymidine. The values are reported as a percentage of parents in the S phase and are normalized for the total number of parents per femur. The results of CFU-GM are shown in Figure 3. The results showed that a single injection of 10 ng per mouse of Exodus significantly reduced the percentage of CFU-GM progenitors in the S phase of the cell cycle to an average of 4 percent (p <0.001), as compared to an average control value of 56 percent CFU-GM in phase S. In addition, the Exodus-induced inhibition of cell cycle progression for CFU-GM was synergistic with treatment with MCP-1 and MIG. When very low concentrations of Exodus were injected with MIG or MCP-1, there was even greater reduction of CFU-GM in phase S. Therefore, a combination of chemokines can ^ P * produce a more potent inhibition of hematopoiesis. These results indicate that the Exodus can temporarily stop the cycle advance of the cell the bone marrow progenitor cells, therefore can be used to protect the normal bone marrow against cytotoxic chemotherapy of the S phase.

B. Effect on the Lines of the Myeloid Cell The effect of the Exodus protein products on the proliferation of myeloid cell lines that depend on cytokine was also tested. The human myeloid cell lines TF-1 and M07E [Avanzi et al., Bri t. J. > Haematol , 69: 359 (1988)] require GM-CSF and SCF for maximum proliferation. The primitive acute myeloid leukemia cell lines depending on the cytokine TF-1 and M07E (both gifts from Dr. Hal Broxmeyer, Indiana University, Indiana) were grown in RPMI 1640 (Life Technologies, Gaithersburg, MD) plus 10 percent of FCS (Hyclone) and 100 units per milliliter of penicillin and 100 micrograms per milliliter of streptomycin (tissue culture antibiotics, Life. Technology, Gaithersburg, MD). This medium is supplemented with the granulocyte-macrophage colony stimulation factor (GM-SCF, 100 units per milliliter, Immunex, Seatle, WA) and the hemocytoblast factor (SCF, 50 ng / milliliter, Amgen, Thousand Oaks, CA ) for the growth of the normal logarithmic phase. Exodus in the supernatants of the cell COS prepared as described in Example 3 was tested at a final dilution of 1/10. The results are shown in Figure 4 (for M07E cells) and in Figure 5 (for TF-1 cells). When the supernatant of the COS cells was added to the M07E cells of the logarithmic phase in the presence of GM-CSF and SCF, the proliferation over the following 72 hours was reduced to 10.4 percent control. When the COS cell supernatant fluid was added to the TF-1 cells of the logarithmic phase which were also continuously exposed to GM-CSF and SCF, the proliferation was completely inhibited. Exodus was not exerting a cytotoxic effect, since cells treated with Exodus had greater viability than # 95 percent at each point in time, as evaluated by the exclusion of trypan blue, which was identical to that of the control cells. The purified synthetic Exodus also completely inhibited the proliferation of M07E cells as shown in Figure 6. Each data point is the means of four separate experiments. The stars represent the statistical significance in p < 0.05 using a t test in ^? couple The viability also did not change with the addition of Exodus. These results indicate that Exodus will also be useful for treating myeloproliferative disorders such as chronic myelogenous leukemia C. Effect on the Progenitors of Chronic Myelogenous Leukemia The effect of Exodus (also called "Exodus-1") on progenitor proliferation in chronic myelogenous leukemia (CML) was evaluated using colony formation assays as described in the Hromas article. and others, Blood, 89: 3315-3322 (1997). By abbreviating, the bone marrow cells were collected in six CML patients in the chronic phase. The cells of the low density marrow at 5 x cells per milliliter were placed in - 1 percent of ilcellulose in the modified Dulbecco's medium of Iscove supplemented with 30 percent fetal calf serum, 1 unit per milliliter of human erythropoietin (Epogen®, Amgen), 100 units per milliliter of human interleukin-3 (Genetics Institute) and 50 ng per milliliter of the factor of human ocitoblast (Amgen) in the presence or absence of 100 ng per milliliter of Exodus and in the presence or absence of 100 ng per milliliter of MlP-la. The cultures were incubated at 5 percent CO2 and low oxygen tension (5 percent) for 14 days, and then classified using an inverted microscope for CFU-GM, CFU-GEMM and BFU-E. The colony counts for cultures treated with Exodus or MlP-la were compared with the colony counts of the control cultures and expressed as a percentage of CFU or BFU control. The data is disclosed in Table 3, which is presented below. 25 Table 3 Treatment% Control% Control% Control CFU-GM BFU-E CFU-GEMM 100 ng / milliliter 52 + 5 ^ 44 + 19 '57 + 6 ^ of Exodus 100 ng / milliliter of MlP-la + 3 1 + 3 + 6 * Statistically significant (p <0.05) using Student's t-test not in pairs.

These data demonstrate that in these six patients with CML in the chronic phase, Exodus markedly inhibited the formation of the progenitor colony. Exodus was much more effective than MlP-la in suppressing proliferation by suggesting that the effects of Exodus are mediated by a receptor that MlP-la does not activate. The progenitors of CML overexpress in BCR-ABL fusion coprotein, a constitutively activated cytoplasmic tyrosine kinase that stimulates proliferation. In fact, the forced over-expression of BCR-ABL in the cell culture is transformed into many cell types including NIH 3T3 cells. The effect of # Exodus on the progression of the progenitor cell cycle of three chronic phase CML patients was further explored using a lithiated thymidine killing assay as described above. Exodus treatment of CML progenitors halted cell cycle progression by an average of 55 + 5 percent CFU-GM, 45 + 13 percent BFU-E, and 50 + 10 percent CFU- GEMM In this way, the Exodus inhibitory signal was able to overcome the aggressive proliferative signal of BCR-ABL in the 10 progenitors of CML. These results indicate that Exodus suppresses hematopoiesis and may be effective in treating CML in the chronic phase.

EXAMPLE 11 Effect of Exodus on HIV Proliferation # The ability of Exodus protein products to inhibit HIV proliferation as measured by The HIV production of the p24 protein was tested using a standard p24 ELISA as described previously in Cocchi et al., Science, 270: 1811 (1996). Human peripheral blood mononuclear cells from a normal volunteer were isolated on a Ficoll gradient. These cells were activated with 1 ng per milliliter of PHA (Sigma, St. !5 - Louis, MO) in RPMI 1640 (Life Technologies, Gaithersburg, MD) plus 10 percent FCS (Hyclone) and 100 units per milliliter of penicillin and 100 micrograms per milliliter of streptomycin (tissue culture antibiotics, Life Technologies, Gaithersburg, MD) for 48 hours at 37 ° C, washed in a complete medium, and then infected with TCID5o = 5000 of the HIV strains designated BAL (from ATCC) or A018-H112-2 (from ATCC) for 1 hour in a complete medium at 37 ° C. The cells were then washed three times in the medium to remove the excessive virus, and resuspended at 5 x 10-5 cells / 0.3 milliliter per test in a complete medium plus recombinant human IL-2 (10 ng per milliliter, Boehringer-Maim, Indianapolis, IN) plus recombinant Exodus or the supernatants of the COS cell tranfected with pECE as controls. After six days of culture, the cell-free supernatant liquids were tested for their p24 HIV content using an enzyme-linked immunosorbent assay (ELISA, ELISA, Abbott Laboratories, Chicago, IL). The experiments were carried out in triplicate. The supernatant fluid of the COS cell containing Exodus at a final dilution of 1: 2 (ie, 0.15 milliliter of COS cell supernatant in 0.3 milliliter in total in each well), the supernatant fluid of the COS cell containing Exodus at a final dilution of 1: 4, MlP-la at 625 ng / milliliter and 1250 ng / milliliter (bar 1 and bar 2, respectively, in Figure 7) and the supernatant the COS cell pECE (without Exodus) were measured 6 days after infection. The results are shown in Figure 7. When normal human peripheral blood mononuclear cells stimulated by PMA were infected at high multiplicity with two strains of HIV, Exodus was able to significantly inhibit the proliferation of HIV in both strains. At the highest concentration of the recombinant Exodus used, the proliferation of HIV BAL strain was decreased up to 39 percent of the control while the proliferation of HIV from strain A018 was reduced up to 27 percent control. This inhibition was less noticeable when the concentration of Exodus was reduced. In addition, this inhibition was compatible with that seen with MlP-the one that was used as a positive control in these experiments. The inhibition by Exodus was not due to phytotoxicity since there is no difference in the viability of the cells treated with Exodus as with the control cells. Similar results are shown in Figure 8 for a purified synthetic Exodus protein product (Exodus with additional alanine after residue 4) to a concentration of 1 microgram per milliliter and MlP-la to a 7 - # concentration of 1 microgram per milliliter. The results are shown at 3, 6 and 9 days after infection. At 9 days after infection, the inhibitory effect of Exodus was similar to that seen with MlP-la at the same concentration. In this preliminary experiment, no effects are seen with this Exodus protein product at concentrations less than 1 microgram per milliliter. * These results indicate that the products of Exodus protein inhibits the proliferation of HIV, and therefore will be useful in method for increasing resistance to HIV infection and methods to treat HIV infection.

HIV EXAMPLE 12 Assay of the Chemoattractant Properties and # Activation of Exodus Cells in Human Monocytes / Human Macrophages and Neutrophils The effects of Exodus on human monocytes / human macrophages or neutrophils is evaluated, v.gr, by the methods described by Devi et al., J. Immunol., 153: 5376-5383 (1995) to evaluate the activation induced by TCA3- Murine of neutrophils and macrophages.

The activation rates measured in these studies include # increased adhesion to fibrinogen due to integrin activation, chemotaxis, induction of reactive nitrogen intermediates, respiratory burst (production of superoxide and hydrogen peroxide), and exocytosis of lysozyme and elastase in the presence of cytochalasin B. As discussed by Devi and others, these activities correlate with several stages of the leukocyte response to inflation. This leukocyte response, reviewed by Springer, Cell, '76: 301-314 (1994), involves adhesion of leukocytes to endothelial cells of blood vessels, migration through the endothelial layer, chemotaxis towards a source of chemokines, and site-specific release of inflammatory mediators. The Exodus involved in any of these stages provides an important target for clinical intervention by modulating the inflammatory response.

EXAMPLE 13 Exodus In Vivo Tumor Growth Inhibition Assay The growth inhibition properties of the Exodus tumor were tested, e.g., by modifying the protocol described by Laning et al. J. Immunol, 153: 4625-4635 (1994) to test the inhibitory properties. of murine TCA3 tumor growth. An Exodus coding cDNA was transfected by electrophoration into the cell line derived from myeloma J558 (American Type Culture Collection, Rockville, MD). The transfectants were selected for Exodus production by standard techniques such as ELISA (enzyme-linked immunosorbent assay) using a monoclonal antibody generated against Exodus as detailed in Example 8. A large ball of 10 million cells from an Exodus producing clone is injected subcutaneously into the lower right quadrant of BALB / c mice. For comparison, 10 million untransfected cells were injected into a control mouse. The rate and frequency of tumor formation in the two groups is compared to determine the efficacy of Exodus to inhibit the tumor's credibility. The nature of the cellular infiltrate subsequently associated with the tumor cells is identified histologically. In addition, the recombinant Exodus (20 ng) is mixed with untransfected J558 cells and injected (20 ng per day) into the tumors derived from these cells, to test the effect of Exodus administered exogenously on the tumor cells.

EXAMPLE 14 Intraperitoneal Injection Assay Cells that respond to Exodus in vivo were determined by injection of 1 to 100 ng of purified Exodus into the intraperitoneal cavity of the mice, as described by Luo et al., J. Immunol, 153 : 4616-4624 (1994). After injection, the leukocytes were isolated from the peripheral blood and the peritoneal cavity and identified by staining with the Diff Quick kit (Baxter, McGraw, IL). The profile of Leukocytes are measured several times to evaluate the kinetics of the appearance of different cell types. In separate experiments, neutralization antibodies directed against Exodus (Example 8) are injected together with Exodus to confirm that the Leukocyte infiltration is due to the activity of Exodus. # EXAMPLE 15 In Vivo Activity Test - Subcutaneous Injection 20 The chemoattractant properties of Exodus are tested in vivo by adapting the protocol described by Meurear et al., J. Exp. Med., 178: 1913-1921 (1993). The recombinant Exodus (10-500 pmol / site) is injected intradermally towards an appropriate mammal, e.g., dogs or rabbits. During the times of 4 to 24 hours, the infiltration of the cell at the site of injection is assessed by histological methods. The presence of Exodus is confirmed by immunocytochemistry using antibodies directed against Exodus. The nature of the cellular infiltrate is identified by dyeing with the Baxter Diff Quick kit.

EXAMPLE 16 10 Cloning an Exodus Receptor The DNA encoding an Exodus receptor is cloned by adapting the procedures described above for the isolation of the IL-8 receptor gene in Holmes et al. above, and the isolation of the MCP-1 receptor gene in Charo et al., Supra. A cDNA library is preferably prepared from cells that respond to Exodus by chemotaxis and activation. The irradiated Exodus can also be used to identify cell types that express high levels of the receptor for Exodus. Cells that do not respond to MIP-1 a or RANTES or cells that show a different pattern of desensitization to the receptor in response to these coordinating groups are of specific interest. The puddles of clones transfected into the cDNA library are selected # for irradiated Exodus binding by autoradiography. The positive pools are successively sub-divided and re-selected until the individual positive clones are obtained. Alternatively, a degenerate PCR strategy can be used wherein the sequences of the PCR primers are based on conserved regions of the known chemokine receptor sequences. For # increase the possibility of isolating an Exodus receptor, DNA used in the reaction may be cDNA derived from a cell type that responds to Exodus. Although the present invention has been described in terms of specific modalities, it will be understood that variations and modifications will occur to them. those people skilled in the art. Accordingly, only those limitations appearing in the appended claims should be included in the invention.

F SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Indiana University Foundation (ii) TITLE OF INVENTION: 5 EXODUS CHEMICALS AND MATERIALS (iii) NUMBER OF SEQUENCES: 4 (iv) ADDRESS FOR CORRESPONDENCE: # ( A) RECIPIENT: Marshall, O'Toole, Gerstein, Murray & Borun 10 (B) STREET: 233 South Wacker Drive / 6300 Sears Tower (C) CITY: Chicago (D) STATE: Illinois (E) COUNTRY: United States of America 15 (F) POSTAL CODE: 60606-6402 (v) FORM READ IN COMPUTER: (A) TYPE OF MEDIUM: Diskette (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS 20 (D) FOSTWARE: Patent in Release # 1.0, Version # 1.30 ( vi) CURRENT REQUEST DATA: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: 25 (C) CLASSIFICATION: 'H (vii) APPLICATION DATA - ABOVE: (A) APPLICATION NUMBER: US 08 / 749,513 (B) SUBMISSION DATE: NOVEMBER 15, 1996 (viii) LAWYER / AGENT INFORMATION: (A) NAME: Rin-Laures, Li-Hsien (B) REGISTRATION NUMBER: 33,547 (C) REFERENCE / NUMBER OF TOUCH OF ATTORNEY # 27866/34328 PCT 10 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (312) 474-6300 (B) TELEFAX: (312) 474-0448 (2) INFORMATION FOR SEQ ID NO: l: 15 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 821 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear 20 (ii) TYPE OF MOLECULE: DNA (genomic) (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 43..327 (ix) PARTICULARITY: 25 (A) NAME / KEY: mat peptide * (B) LOCATION: 109. .327 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l: GGTACTCAAC ACTGAGCAGA TCTGTTCTTT GAGCTAAAAA CC ATG TGC TGT ACC 54 Met Cys Cys Thr-22 -20 AAG AGT TTG CTC CTG GCT GCT TTG ATG TCA GTG CTG CTA CTC CAC CTC 102 Lys Ser Leu Leu Leu Ala Ala Leu'Met Ser Val Leu Leu Leu His Leu -15 -10 -5 TGC GGC GAA TCA GAA GCA AGC AAC TTT GAC TGC TGT CTT GGA CT ACA 150 Cys Gly Glu Ser Glu Wing Ser Asn Phe Asp Cys Cys Leu Gly Tyr Thr 10 1 5 10 GAC CGT ATT -CTT CAT CCT AAA TTT ATT GTG GGC TTC ACA CGG CAG CTG 198 Asp Arg lie Leu His Pro Lys Phe He Val Gly Phe Thr Arg Gln Leu 15 20 25 30 GCC AAT GAA GGC TGT GAC ATC AAT GCT ATC ATC TTT CAC ACA AAG AAA 246 Wing Asn Glu Gly Cys Asp He Asn Wing He He Phe His Thr Lys Lys 35 40 45 AAG TTG TCT GTG TGC GCA AAT CCA AAA CAG ACT TGG GTG AAA TAT ATT 294 Lys Leu Ser Val Cys Ala Asn Pro Lys Gln Thr Tp Val Lys Tyr He 15 50 55 60 GTG CGT CTC CTC AGT AAA AAA GTC AAG AAC ATG TAAAAACTGT GCCTTTTCTG 347 Val Arg Leu Leu Ser Lys Lys Val Lys Asn Met 65 70. GAATGGAATT GGACATAGCC CAAGAACAGA AAGAACCTTG CTGGGGTTGG AGGTTTCA CT 407 TGCACATCAT GGAGGGTTTA GTGCTTATCT AATTTGTGCC TCACCTGGAC TTGTCCAATT 467 AATGAAGTTG ATTCATATTG CATCATAGTT TGCTTTGTTT AAGCATCACA TTAAAGTTAA 527 ACTGTATTTT ATGTTATTTA TAGCTGTAGG TTTTCTGTGT TTAGCTATTT AATACTAATT 587 TTCCATAAGC TATTTTGGTT TAGTGCAAAG TATAAAATTA TATTTGGGGG GGAATAAGAT 647 TATATGGACT TTCTTGCAAG CAACAAGCTA TTTTTTAAAA AAAACTATTT AACATTCTTT 707 TGTTTATATT GTTTTGTACT CCTAAATTGT TGTAATTGCA TTATAAAATA AGAAAAATAT 767 TAATAAGACA AATATTGAAA ATAAAGAAAC AAAAAGTTCT TCTGTTAAAA AAAA. 821 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 95 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: pro tein (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Met Cys Cys Thr Lys Ser Leu Leu Leu Ala Wing Leu Met Ser Val Leu -. 10 - 22 -20 - 15 -10 Leu Leu Hie Leu Cys Gly Glu Ser Glu Wing Being Asn Phe Asp Cye Cye -5 1 5 10 Leu Gly Tyr Thr Aep Arg He Leu His Pro Lys Phe He Val Gly Phe 15 20 25 Thr Arg Gln Leu Wing Asn Glu Gly Cys Asp He Asn Wing He ne Phe 35 40 Trp (2) INFORMATION FOR SEQ ID NO: 3: 20 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear 25 (ii) TYPE OF MOLECULE: nucleic acid * (xi) DESCRIPTION OF THE SEQUENCE: "SEQ ID NO: 3: GGCGAAGCTT TGAGCTAAAA ACCATG 26 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid 10 (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: GCGGGAATTC TTACATGTTC TTGACT '26 ?

Claims (9)

f ^^^ R E I V I N D I C A C I O N S

1. A purified polynucleotide encoding the Exodus amino acid sequence of SEQ ID NO:

2. 2. The polynucleotide of claim 1, which is a DNA.

3. The DNA of claim 2 comprising a nucleotide sequence consisting of nucleotides

4. A purified polynucleotide encoding amino acids 1 to 73 of SEQ ID NO: 2.

5. The polynucleotide of claim 4 which is a DNA

6. The DNA of claim 5 comprising a nucleotide sequence consisting of nucleotides r comprising the DNA of claim 2, 3, 5 or 6. # 8. The vector of claim 7 which is an expression vector, wherein the DNA is operably linked to the expression control DNA sequence. 9. A transformed host cell stably transfected with the DNA of claim 2, 3, 5 or 6, so as to allow expression in the Exodus host cell. A method for producing Exodus which comprises culturing the host cell in claim 9 in a nutrient medium and isolating the Exodus from the host cell or the nutrient medium. 11. A purified polypeptide produced by the method of claim 10. 12. A purified polypeptide comprising the Exodus amino acid sequence of SEQ ID NO: 2. 13. A purified polypeptide comprising amino acids Exodus 1 to 73 of SEQ ID NO: 2. 14. A hybridoma cell line that produces a monoclonal antibody that is specifically reactive 15 with the polypeptide of claim 15. 15. The monoclonal antibody produced by the hybridoma of claim 14. 16. A method for increasing the resistance to infection of the human immunodeficiency virus (HIV) that 20 comprises administering to a patient an amount of the effective Exodus protein product to inhibit the proliferation of HIV. 1

7. The method of claim 16, wherein the patient is at risk of being exposed to HIV, has been 25 exposed to HIV or has been infected with HIV. * 1

8. A method for treating human immunodeficiency virus (HIV) infection which comprises administering to an HIV-infected patient an amount of the Exodus protein product effective to inhibit the proliferation of HIV. 1

9. The use of Exodus protein product in the preparation of a medicament for the prophylactic or therapeutic treatment of HIV infection. 20. The use according to claim 10 19, wherein the medicament inhibits the proliferation of HIV 21. A method for protecting the progenitor cells of the bone marrow from the cytotoxic effects comprising the administration to a patient of a The amount of the Exodus protein product effective to suppress the proliferation of the progenitor cell of the ^ F bone marrow. 22. The method of claim 21, wherein the patient undergoes chemotherapy or radiotherapy. 23. The use of the Exodus protein product in the preparation of a medicament for the suppression of the proliferation of the bone marrow progenitor cell. The method of claim 23, wherein the medicament is administered to a patient undergoing treatment by chemotherapy or radiotherapy. 25. A method for treating myeloproliferative diseases comprising administering to a patient suffering from a myeloproliferative disease, an amount of an Exodus protein product effective to suppress the proliferation of the progenitor cells of the malignant bone marrow. 26. The method of claim 25, wherein the myeloproliferative disease is leukemia, chronic myelogenous, essential thrombocytosis, myelofibrosis or polycythemia vera. 27. The use of the Exodus protein product in the preparation of a drug for the treatment of 15 myeloproliferative diseases. 28. The use according to claim 27, wherein the myeloproliferative disease is chronic myelogenous leukemia, essential thrombocytosis, myelofibrosis or polycythemia vera.