KR19990008335A - Human Chemokine Beta-8, Chemokine Beta-1, and Macrophage Inflammatory Protein-4 - Google Patents

Abstract

The present invention relates to DNA (RNA) encoding human Ckβ-8, MIP-4 and Ckβ-1 and chemokine polypeptides, and a method for producing the polypeptide by recombinant technology is disclosed. Also disclosed are methods of using chemokine polypeptides for leukemia, tumors, chronic infections, autoimmune diseases, fibrotic diseases, wound healing and psoriasis. The use of antagonists against chemokine polypeptides and antagonists against rheumatoid arthritis, autoimmune and chronic and acute inflammatory and infectious diseases, allergic reactions, postglandin-dependent fever and osteomyelitis. Also disclosed are diagnostic assays for detecting concentration changes in diseases and polypeptides associated with variations in nucleic acid sequences.

Description

Human Chemokine Beta-8, Chemokine Beta-1, and Macrophage Inflammatory Protein-4

Chemokines, called interclean cytokines, are a subset of cytokines that are structurally and functionally related. These molecules are 8-10 kd in size. Generally, chemokines exhibit 20% to 75% homology at the amino acid level and are characterized by four conservative cysteine residues that form two disulfide bonds. Chemokines were classified into two subgroups, alpha and beta, based on the arrangement of the first two cysteine residues. In the alpha subgroup, the first two cysteines are separated by one amino acid and are therefore also referred to as C-X-C subgroups. In the beta subgroup, two cysteines are adjacent to each other and are therefore called the C-C subgroup. To date, more than eight different components of this group have been identified in the human body.

Interclean cytokines exhibit a variety of functions. Its unique feature is its ability to attract chemotactic migration of unique cell types, including monocytes, neutrophils, T lymphocytes, basophils and fibroblasts. Many chemokines have proinflammatory activity and are involved in multiple stages during the inflammatory response. These activities include histamine release stimulation, lysosomal enzyme and leukotriene release, increased adhesion of target immune cells to endothelial cells, increased binding of complement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory explosions. do. In addition to its relevance to inflammation, certain chemokines have been shown to exhibit different activities. For example, macrophage inflammatory protein 1 (MIP-1) can inhibit proliferation of hematopoietic hepatic cells, platelet factor-4 (PF-4) is a potent inhibitor of endothelial cell proliferation, and interleukin-8 (IL -8) promotes proliferation of keratinocytes and GRO is an autocleaning factor for melanoma cells.

In view of its various biological activities, it is not surprising that chemokines are involved in many physiological and disease states, including lymphocyte migration, wound healing, hematopoietic control, and inflammatory diseases such as allergies, asthma and arthritis. One example of a hematopoietic regulator is MIP-1. MIP-1 was originally identified as an endotoxin-induced proinflammatory cytokine produced from macrophages. Subsequent studies revealed that MIP-1 consists of two different but related proteins MIP-1α and MIP-1β. MIP-1α and MIP-1β are chemotactic factors for macrophages, monocytes and T lymphocytes. Interestingly, biochemical purification and serial sequencing of pluripotent liver cell inhibitors (SCI) revealed that SCI was identical to MIP-1α. Moreover, MIP-1β may react with the property of MIP-1α to inhibit hematopoietic liver cell proliferation. These findings lead to the hypothesis that the primary physiological role of MIP-1 is to regulate hematopoiesis in the bone marrow and that the proposed inflammatory action is secondary. The mode of action of MIP-1α as a hepatic cell inhibitor is associated with the blocking of the cell cycle in the G1 / S mitotic phase. In addition, the inhibitory effect of MIP-1α is limited to immature progenitor cells but is presumed to be substantially stimulating to late progenitor cells in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF).

Several groups cloned the potential for human homologues of MIP-1α and MIP-1β. In all cases, cDNA was isolated from the library prepared for activated T-cell RNA.

MIP-1 protein can be detected in inflammatory cells in early wounds and has been shown to induce the production of IL-1 and IL-6 from fibroblasts in wounds. In addition, purified natural MIP-1 (containing MIP-1, MIP-1α and MIP-1β polypeptides) causes acute inflammation when injected intracutaneously or into the cerebrospinal fluid of rabbits (Wolpe and Cerami, 1989). , FASKB J. 3: 2565-73). In addition to the proinflammatory properties of MIP-1, which may be of direct or indirect nature, MIP-1 was also collected at the early inflammatory stage of wound healing in experimental mouse models using sterile wound treatment chambers (Fahey et al., 1990, Cytokine, 2:92). For example, PCT application WO 92/05198 filed by Chiron Corporation discloses an active DNA molecule as a template for producing mammalian macrophage inflammatory protein (MIP) in yeast.

MIP-1α and MIP-1β in rats are distinct but closely related cytokines. A partially purified mixture of these two proteins affects neutrophil function and causes local inflammation and fever. MIP-1α was expressed in yeast cells and purified to homogenates. As a result of structural analysis, it was confirmed that MIP-1α has a secondary structure and a tertiary structure very similar to PF-4 and IL-8, which share limited sequence homology. In addition, MIP-1α has been shown to be active in vivo to inhibit mouse liver cells in vitro killing by continuous tritiated thymidine treatment. In addition, MIP-1α has been shown to enhance proliferation of more antigen-sensitized progenitor granulocyte macrophage colony-forming cells in response to granulocyte macrophage colony-stimulating factors (Clemens, JM, et al., Cytokine, 4 :). 76-82 (1992).

This application is part of a continuation of pending application 08 / 446,881, filed May 5, 1995 with the United States Patent and Trademark Office.

The present invention relates to newly identified polynucleotides, polypeptides encoded by these polynucleotides, uses of these polynucleotides and polypeptides, and methods of preparing these polynucleotides and polypeptides. More specifically, the polypeptides of the present invention are assumed as human chemokine β-8 (Ckβ-8), macrophage inflammatory protein-4 (MIP-4) and chemokine β-1 (Ckβ-1). The invention also relates to a method of inhibiting the action of such polypeptides.

The accompanying drawings are not intended to limit the scope of the invention as defined by the claims.

1 shows a cDNA sequence encoding Ckβ-8 and the corresponding deduced amino acid sequence. The first 21 amino acids represent the putative leader sequence. The signal sequences are all as determined by N-terminal peptide sequencing of Baculovirus expressing proteins.

2 shows a cDNA sequence encoding Ckβ-1 and its corresponding deduced amino acid sequence. The first 19 amino acids represent the leader sequence.

Figure 3 shows the cDNA sequence encoding MIP-4 and the corresponding deduced amino acid sequence. The first 20 amino acids represent the leader sequence.

4 illustrates amino acid homology between Ckβ-8 (top) and human MIP-1α (bottom). Four cysteines characteristic of all chemokines are shown.

5 shows two amino acid sequences, the top sequence is the human MIP-4 amino acid sequence and the bottom sequence is the human MIP-1α (human amygdala lymphocyte LD78 beta protein precursor).

Figure 6 shows the alignment of amino acid sequences between Ckβ-1 (top) and human MIP-1α (bottom).

7 is a gel photograph of Ckβ-1 electrophoresed after expression of HA-tagged Ckβ-1 in COS cells.

8 is a gel photograph of SDS-PAGE after expression and purification of Ckβ-1 in Baculovirus expression system.

9 is a gel photograph of SDS-PAGE after Ckβ-8 expression and three steps purification in a Baculovirus expression system.

10A and 10B show chemotactic activity of Ckβ-8 measured by chemotaxis assay using 48-well microchamber instrument (Neuro Probe, Inc.). The experimental procedure was performed as described in the manufacturer's manual. For each concentration of Ckβ-8 tested, the mobility at high power field of 5 was investigated. The results presented represent the mean values obtained in each of the two experiments. Chemotactic activity against THP-1 (A) cells and human PBMC (B) is shown.

Fig. 11 shows changes in intracellular calcium concentration in response to Ckβ-8 measured using a Hitachi F-2000 fluorescence spectrophotometer. Bacterial expressing Ckβ-8 was added to Indo-1 loaded THP-1 cells at a final concentration of 50 nM and the intracellular concentration of calcium concentration was monitored.

FIG. 12 shows that the mononuclear cell line THP-1 was treated with LPS (0.1-10 ng / ml) or Ckβ-8 (50 ng / ml) for 16 hours, followed by ELISA analysis of the supernatant of the tissue culture to determine TNF-α secretion. It is quantified.

FIG. 13A and FIG. 13B show that the human peripheral blood monocytes purified by the elution method were treated with an increased amount of Ckβ-8 (product of Baculovirus) for 16 hours, and then the tissue culture supernatant was subjected to ELISA analysis for TNF-α and IL-. 6, IL-1, GM-CSF and granulocyte-colony stimulating factor (G-CSF) secretion amount was quantified.

FIG. 14 shows a low density population of mouse bone marrow cells, IL-3 (5ng / ml), SCF (100ng / ml), IL-1α (10ng) in agar medium with or without the indicated chemokine (100ng / ml). / ml) and plated in the presence of M-CSF (5ng / ml) (1,500 cells / dish). The data presented represent the mean values obtained in each of the two experiments (two repeated each). Colonies were counted 14 days after plate culture. The number of colonies produced in the presence of chemokines is expressed as the average number of colonies produced in the absence of any chemokines added.

15A and 15B show the effects of Ckβ-8 and Ckβ-1 on mouse bone marrow colony formation represented by HPP-CFC (A) and LPP-CFC (B).

FIG. 16 illustrates the effect of Baculovirus-expressing Ckβ-1 and Ckβ-8 on M-CFS and SCF-stimulated colony formation in freshly isolated bone marrow cells.

FIG. 17 depicts the effects of Ckβ-8 and Ckβ-1 on IL-3 and SCF-stimulated proliferation and differentiation of cognate populations of bone marrow cells.

18A and 18B depict the effects of Ckβ-8 and Ckβ-1 on the generation of GR-1 and Mac-1 (surface marker) positive cell populations among cognate populations of bone marrow cells. Cognate cells were grown with supplementation medium (a) supplemented with IL-3 (5 ng / ml) and SCF (100 ng / ml) alone and with supplementation with Ckβ-8 (50 ng / ml) or Ckβ-1 (50 ng / ml). Incubated in medium (b). Cells were then stained using monoclonal antibodies against myeloid differentiation GR.1, Mac-1, Sca-1 and CD45R surface antigens and analyzed by FACScan. Data are presented as percentage (%) of positive cells in large cell population (A) and small cell population (B).

FIG. 19 illustrates that the presence of Ckβ-8 (+) inhibits bone marrow cell colony formation in response to IL-3, M-CSF and GM-CSF.

20 is a dose response response of Ckβ-8 that inhibits bone marrow cell colony formation. Cells were isolated and treated as in FIG. 19. The cells thus treated were treated with IL-3, GM-CSF or M-CSF (5 ng) with or without Ckβ-8 at 1 ng / ml, 10 ng / ml, 50 ng / ml and 100 ng / ml. / ml) and plated at a density of 1,000 cells / dish in an agar colony formation assay. The data show colony formation rate as% colony formed by the specific factor alone. This data is presented as the average value of the duplicate culture dish with error bars indicating standard deviation.

21 depicts apoptosis induction responses by Ckβ-8 and Ckβ-1 in the presence or absence of hematopoietic growth factor. Mouse bone marrow cells were perfused in the femur and tibia and separated with a Ficoll density gradient, followed by the release of monocytes by plastic adhesion. The obtained cell population was then added with or without Ckβ-8 (50ng / ml) or Ckβ-1 (250ng / ml) IL-3 (5ng / ml), GM-CSF (5ng / ml), M- Incubated overnight in MEM-based medium supplemented with CSF (10 ng / ml) and G-CSF (10 ng / ml). In addition, cells were cultured in medium alone with or without Ckβ-8 and Ckβ-1. After 24 hours, cells were harvested and treated with an apoptosis reaction using the Schöllinger Mannheim cell death ELISA kit. The data are expressed as growth rates above this threshold using a baseline value that is considered to be the amount of apoptosis present in culture cultured in the presence of each growth factor.

Figure 22 shows the expression rate of RNA encoding Ckβ-8 in human monocytes. Total RNA was isolated from freshly eluted monocytes and treated with hu rIFN-g 100 U / ml, LPS 100 ng / ml or mixtures thereof. RNA after each treatment was separated by electrophoresis on 1.2% agarose gel and transferred to nylon membrane. Ckβ-8 mRNA was quantified by probe with ³² P-labeled cDNA, and the band on the obtained autoradiograph was quantitatively quantitatively quantified.

According to one aspect of the invention, a mature polypeptide having the deduced amino acid sequence of FIGS. 1, 2 and 3 (SEQ ID NOS: 2, 4 and 6, respectively) or a clone of ATCC Accession No. 75676 deposited February 9, 1994 Mature Ckβ-8 polypeptide encoded by cDNA of (s), and mature MIP-4 polypeptide encoded by cDNA of clone of ATCC Accession No. 75675 deposited February 9, 1994, and October 13, 1993 An isolated nucleic acid (polynucleotide) is provided that encodes a mature Ckβ-1 polypeptide encoded by the cDNA of a clone of deposited ATCC accession number 75572.

Polynucleotides encoding polypeptides of the invention are structurally related to interclean, a pro-inflammatory supergene belonging to cytokines or chemokines. Ckβ-8 and MIP-4 are both MIP-1 homologues and have more homology to MIP-1α than MIP-1β. The polynucleotide encoding Ckβ-8 is derived from the aortic endothelial cDNA library and contains an open reading frame encoding a polypeptide of 120 amino acid residues that exhibits significant homology with many chemokines. The upper registration rate is for human macrophage inflammatory protein 1α showing 36% homology and 66% similarity (FIG. 4).

Polynucleotides encoding MIP-4 are derived from human adult lung cDNA libraries and contain an open reading frame encoding polypeptides of 89 amino acid residues that show significant homology with many chemokines. The upper registration rate is for human tonsil lymphocyte LD78β protein, showing 60% homology and 89% similarity (FIG. 5). In addition, the four cysteine residues present in all chemokines among the characteristic motifs are conserved in both clone (s). Due to the fact that the first two cysteine residues in these genes are in adjacent positions, these genes are classified as C-C or β subgroups of chemokines. One amino acid is mediated between the first two cysteine residues in another subgroup, CXC or α subgroup.

The polynucleotide encoding Ckβ-1 contains an open reading frame that encodes a polypeptide consisting of 93 amino acids in which the first 19 are leader sequences and the mature polypeptide contains 74 amino acid residues. Ckβ-1 shows significant homology to the human macrophage inflammatory protein α, with 48% homogeneity and 72% similarity for the 80 amino acid stretch. In addition, the four cysteine residues that make up the characteristic motif are also conserved.

Polynucleotides of the invention may be in the form of RNA or DNA, and DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double stranded or single stranded, and the single stranded strand may be a coded strand or a non-coding (antisense) strand. The coding sequence encoding the mature polypeptide is the same coding sequence as shown in FIGS. 1, 2 and 3 (SEQ ID NOS: 1, 3, and 5, respectively) or the coding sequence of the deposited clone (s), or the coding sequence is depicted. It encodes the same mature polypeptide as DNA of 1, 2 and 3 (SEQ ID NOS 1, 3 and 5, respectively) or deposited cDNA (s) but may be different coding sequences due to the abundance or degeneracy of the genetic code.

Polynucleotides encoding the mature polypeptide of FIG. 1, FIG. 2 and FIG. 3 (SEQ ID NOS: 2, 4 and 6) or the mature polypeptide encoded by deposited cDNA (s) may comprise the coding sequence of the mature polypeptide; Coding sequences of mature polypeptides and additional coding sequences such as leader or secretory sequences or shear protein sequences; Coding sequences for the mature polypeptide (and any additional coding sequences) and non-coding sequences such as 5 'and / or 3' of the coding sequence or intron of the coding sequence of the mature polypeptide.

Thus, the term polynucleotide encoding a polypeptide includes not only the coding sequence of the polypeptide, but also polynucleotides containing additional coding sequences and / or non-coding sequences.

The invention also relates to fragments, analogs and derivatives of polypeptides having deduced amino acid sequences of FIGS. 1, 2 and 3 (SEQ ID NOS: 2, 4 and 6) or polypeptides encoded by cDNA of deposited clone (s). It relates to variants of the aforementioned polynucleotides that encode. Variants of polynucleotides may be naturally occurring allelic variants of polynucleotides or non-naturally occurring variants of polynucleotides.

Thus, the present invention provides a polynucleotide encoding the same mature polypeptide as encoded by the same mature polypeptide or cDNA of the deposited clone (s) as shown in FIGS. 1, 2 and 3 (SEQ ID NOS: 2, 4 and 6). , As well as variants of the polynucleotide encoding fragments, derivatives or analogs of the polypeptides of FIGS. 1, 2 and 3 (SEQ ID NOS: 2, 4 and 6) or polypeptides encoded by the cDNA of deposited clone (s). It includes. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As noted above, the polynucleotides will have a coding sequence that is a naturally occurring allelic variant of the coding sequence shown in FIGS. 1, 2 and 3 (SEQ ID NOS: 1, 3 and 5) or the coding sequence of the deposited clone (s). Can be. As is known in the art, allelic variants are alternative forms of polynucleotide sequences that may have substitutions, deletions or additions of one or more nucleotides that do not substantially change the function of the encoded polypeptide.

In addition, the present invention allows the coding sequence of a mature polypeptide to be fused to the same reading frame for a polynucleotide sequence that aids the expression and secretion of the polypeptide from the host cell, such as a leader sequence that regulates the transport of the polypeptide from the cell. Polynucleotides that may be used. A polypeptide having a leader sequence is a shear protein and may have a leader sequence that is cleaved by the host cell to form the mature form of the polypeptide. In addition, the polynucleotide may encode a shear protein that is a mature protein plus an additional 5 ′ amino acid residue. Mature proteins with prosequences are shear proteins and are inactive forms of these proteins. Once the precursor sequence is cleaved off, the active mature protein remains.

Thus, for example, the polynucleotide of the present invention may encode a mature protein, or a protein having a precursor sequence, or a protein having both a precursor sequence and a previous sequence (leader sequence).

In addition, the polynucleotides of the present invention may have coding sequences fused in frame to a marker sequence that enables the polypeptide of the present invention to be purified. The marker sequence is a hexahistidine tag supplied by the pQE-9 vector that allows purification of the mature polypeptide fused to the marker if the host is a bacterium, or for example if the host is a mammal such as a COS-7 cell. Hemagglutinin (HA). HA tags correspond to epitopes derived from influenza hemagglutinin protein (Wilson, I. et al., Cell, 37: 767 (1984)).

The present invention also relates to polynucleotides which hybridize to the above-described sequences when at least 50%, preferably 70%, of the sequences are identical. In particular, the present invention relates to polynucleotides that hybridize to the aforementioned polynucleotides under stringent conditions. The term stringent conditions as used herein means that the hybrid occurs only if the homogeneity between the sequences is at least 95%, preferably at least 97%. In a preferred embodiment, the polynucleotide hybridizing to the above-described polynucleotide has a biological function or activity substantially the same as the cDNA of FIGS. 1, 2 and 3 (SEQ ID NOS: 1, 3 and 5) or the mature polypeptide encoded by the deposited cDNA. Encode the polypeptide that holds.

Alternatively, the polynucleotide has a polynucleotide having at least 20 bases, preferably at least 30, more preferably at least 50 bases, which hybridize to the polynucleotide of the present invention while having homogeneity as described above but without activity. It may be a nucleotide. Such polynucleotides can be used as probes of the polynucleotides for recovering the polynucleotides of SEQ ID NOs: 1, 3 and 5, or as diagnostic probes or PCR primers.

The deposit (s) referred to herein are those deposited under the Budapest Treaty for International Approval of Microbial Deposits in the Patent Procedure. These deposits are provided for convenience only to those skilled in the art and are not in accordance with the recognition that deposits are essential under 35 U.S.C § 112. The sequences of the polynucleotides contained within the deposit, as well as the amino acid sequences of the polypeptides encoded by these sequences, are incorporated herein by reference, and serve to mediate if any dispute arises with the sequences set forth herein. A permit may be required to manufacture, use or sell the deposit, but such a permit will not be granted.

The invention further provides amino acid sequences having fragmented amino acid sequences of FIGS. 1, 2 and 3 (SEQ ID NOS: 2, 4 and 6) or encoded by deposited cDNAs, fragments, analogs and derivatives of these polypeptides. It relates to the Ckβ-8, MIP-4 and Ckβ-1 polypeptide having.

The terms fragments, derivatives, and analogs used in connection with the polypeptides of FIGS. 1, 2 and 3 (SEQ ID NOS: 2, 4 and 6), or polypeptides encoded by deposited cDNA, may be used to describe biological functions that are essentially identical to those polypeptides. By polypeptide that retains activity. Thus, analogs include shear proteins that can be activated by cleavage of the shear protein portion to produce an active mature polypeptide.

The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides.

Fragments, derivatives or analogues of the polypeptides of FIGS. 1, 2 and 3 (SEQ ID NOS: 2, 4 and 6), or polypeptides encoded by deposited cDNAs may be (i) one or more amino acid residues are conservative or non-conservative (Preferably a conservative amino acid residue) and the amino acid residue so substituted is unencoded or unencoded by the genetic code, or (ii) one or more amino acid residues contain substituents, or (iii A) a mature polypeptide is fused with another compound, such as a compound that increases the half-life of the polypeptide (e.g., polyethylene glycol), or (iv) the sequence or leader or secretory sequence used for purification of the mature polypeptide or shear protein sequence. Likewise, additional amino acids may be fused to the mature polypeptide. Such fragments, derivatives and analogs will be apparent to those skilled in the art from the teachings herein within the scope of the present application.

The polypeptide of the present invention is preferably provided in a separate form, and more preferably purified by a homogeneous substance.

The term gene or cistron means a fragment of DNA involved in the production of a polypeptide chain; Coding region before and after regions (leader sequence and trailer sequence) as well as the mediation sequence (intron) between individual code fragments (exons).

The term isolated means that a substance is separated from its original environment (eg, if it is a natural environment). For example, naturally occurring polynucleotides or polypeptides present in an animal body are not isolated, and the polynucleotides or DNAs or polypeptides isolated from some or all of the coexisting materials in nature are isolated. Such polynucleotides may be part of a vector and / or the polynucleotide or polypeptide may be part of a composition, and the vector or composition is isolated in that it is not part of the natural environment.

The invention also relates to the production of a polypeptide of the invention using a vector comprising the polynucleotide of the invention, a host cell genetically engineered with the vector of the invention and recombinant techniques.

Host cells are genetically engineered (transduction or transformation or transfection) using the vectors of the invention, which can be, for example, cloning vectors or expression vectors. The vector may be, for example, a plasmid, virus particles, phage, or the like. The genetically engineered host cell may be cultured in a conventional culture medium modified for the activation of promoters, selection of transformants or amplification of Ckβ-8, MIP-4 and Ckβ-1 genes as needed. Culture conditions, such as temperature, pH, and the like, have already been used when using host cells selected for expression and are obvious to those skilled in the art.

Polynucleotides of the invention can be used to prepare polypeptides using recombinant techniques. Thus, the polypeptide sequence can be included in any one of a variety of expression mediators, in particular in a vector or plasmid for polypeptide expression. Examples of such vectors include chromosomal DNA sequences, non-chromosomal DNA sequences and synthetic DNA sequences such as derivatives of SV40; Bacterial plasmids; Phage DNA; Yeast plasmids; And vectors derived from a combination of plasmids and phage DNAs, vaccinia, adenoviruses, chicken pox viruses, and viral DNAs such as Pseudorabies. However, any other plasmid or vector that can replicate and survive within the host cell can also be used.

Suitable DNA sequences can be inserted into the vector in a variety of ways. Generally, the DNA sequence is inserted into suitable restriction endonuclease site (s) known in the art. These and other methods are those that can be easily performed by those skilled in the art.

The DNA sequence in the expression vector is operably linked to suitable expression control sequence (s) (promoter) to induce mRNA synthesis. Representative examples of such promoters include the LTR or SV40 promoter, E. Coli lac or trp, phage lambda P _L promoter and other promoters known to modulate the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosomal binding site and transcription terminator for initiation of translation. In addition, the vector may comprise a sequence suitable for amplifying expression.

In addition, the expression vector is either dihydrate folate reductase or neomycin resistance for eukaryotic cell culture or E. coli. It is preferred to contain genes that provide phenotypic tracers for the selection of transformed host cells such as tetracycline or ampicillin resistance in E. coli.

A vector containing a suitable DNA sequence as well as a suitable promoter or regulatory sequence as described above can transform the host cell and allow the host cell to express the protein.

Examples of suitable hosts include bacterial cells such as E. coli. Collie, Strepto + myces, Salmonella typhimurium; Fungal cells such as yeast; Insect cells, such as Drosophila S2 and Sf9; Adenovirus; Animal cells such as CHO, COS or Bowes melanoma; Plant cells; and the like. Suitable choices of such hosts will be apparent to those skilled in the art from the teachings herein.

In particular, the present invention also encompasses recombinant constructs comprising one or more sequences as broadly described above. Such constructs include vectors such as plasmids or viral vectors in which the sequences of the invention are inserted in the forward or reverse direction. In one preferred aspect, the construct further comprises a regulatory sequence comprising a regulatory sequence, eg, a promoter, operably linked to the sequence. Many suitable vectors and promoters are known to those skilled in the art and are also commercially available. As examples, the following vectors are shown: for bacteria: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pibluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (stratagen ); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). For eukaryotic cells: pWLNEO, pSV2CAT, pOG44. pXT1, pSG (stratagen), pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, other plasmids or vectors can be used as long as they can replicate in the host and survive.

The promoter region can be selected from any desired gene using a CAT (chloramphenicol transferase) vector or a vector carrying other selectable markers. Two suitable vectors are PKK232-8 and PCM7. Examples of specific bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P _R , P _L and trp. Examples of eukaryotic promoters include early CMV, HSV thymidine kinase, early and late SV40, LTR of retrovirus and murine metallothionein-I. Selection of suitable vectors and promoters is well known to those skilled in the art.

In a further aspect, the invention relates to a host cell containing said construct. The host cell may be a higher eukaryotic cell, for example a mammalian cell or a lower eukaryotic cell, for example a yeast cell, and the host cell may be a prokaryotic cell, for example a bacterial cell. Introduction of the construct into the host cell can be performed by calcium phosphate transfection, DEAE-dextran mediated transfection or electrophoresis (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology (1986)).

In a host cell the construct can be used to prepare a gene product encoded by the recombinant sequence in a conventional manner. Alternatively, the polypeptides of the invention can also be synthesized using conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria or other cells under the control of a suitable promoter. The protein can also be prepared using RNA derived from the DNA constructs of the invention using a cell-free translation system. Cloning and expression vectors suitable for use with prokaryotic and eukaryotic cells are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor (1989). .

Transcription of the DNA encoding the polypeptide of the invention by higher eukaryotic cells is increased by inserting an enhancer sequence into the vector. Enhancers are generally about 10 to 300 bp and are cis-acting elements of DNA that act on the promoter and increase its transcription. Examples include the SV40 enhancer at the bp 100 to 270 tail of the replication origin, the polyoma enhancer and the adenovirus enhancer at the tail of the replication origin.

In general, recombinant expression vectors are the origin of replication and selectable markers such as E. coli. Collie and S. Ampicillin resistance genes of the Cerevisiae TRP1 gene and promoters derived from highly expressed genes are included to transform the host and enable transcription of downstream structural genes. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), alpha-factors, acid phosphatase or heat shock proteins and the like. The heterologous structural sequence is assembled in a suitable phage that possesses a translational initiation sequence and a termination sequence, preferably a leader sequence capable of inducing secretion of the translated protein into the periplasmic interval or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein comprising an N-terminal identification peptide that confers desirable properties such as stabilization or simplified purification of the expressed recombinant product.

Expression vectors useful for bacteria are constructed by inserting into the operable translation a suitable translational initiation and termination sequence containing a promoter of action, along with a structural DNA sequence encoding the protein of interest. The vector includes one or more phenotypic selectable markers and origin of replication to ensure the conservation of the vector and amplification in the host as needed. Examples of suitable prokaryotic hosts for transformation are E. coli. Various species belonging to the genus Coli, Bacillus subtilis, Salmonella typhimurium and Pseudomonas, Streptomyces and Staphylococcus, and other prokaryotic cells can also be used as hosts.

Non-limiting examples of bacterial expression vectors can include bacterial origins of replication and selectable markers derived from commercially available plasmids containing the genetic elements of the well-known cloning vector pBR322 (ATCC 37017). Examples of such commercially available vectors include pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotech, Madison, Wisconsin, USA). These pBR322 backbone sites are combined with a suitable promoter and the structural sequence to be expressed. Hereinafter, transformation of a suitable host bacterium, growth of the host bacterium to a suitable cell density, induction by a suitable means (eg, temperature shift or chemical induction) of a selected promoter, and cell culture for an additional period will be described. .

Cells are typically collected by centrifugation, grinding by physical or chemical means and preservation for further purification of the resulting crude extract.

The microbial cells used for expression of the protein can be pulverized by any convenient method, such as freeze-thaw repeated treatment, sonication, mechanical grinding or cell lysis, which methods are known to those skilled in the art. .

In addition, various mammalian cell culture systems can be used to express recombinant proteins. Examples of mammalian expression systems include COS-7 cell line, a monkey kidney fibroblast described in Gluzman, Cell, 23: 175 (1981), and other cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO. , HeLa and BHK cell lines. Mammalian expression vectors include origins of replication, suitable promoters and enhancers, and also include any necessary ribosome binding sites, polyadenylation sites, ligation donor and receptor sites, transcription termination sequences, and 5 'contiguous non-transcribed sequences. DNA sequences derived from SV40 ligation and polyadenylation sites can be used to provide the necessary nontranscribed genetic elements.

Ckβ-8, MIP-4 and Ckβ-1 are ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography And recovery from recombinant cell culture by a method comprising lectin chromatography. If necessary, protein refolding steps can be used to complete the placement of the mature protein. Finally, high performance liquid chromatography (HPLC) can be used to perform the final purification step.

Polypeptides of the invention may be natural purified products or chemically synthesized products or products produced by recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects and mammalian cells in culture). . Depending on the host used in the recombinant production technique, the polypeptides of the invention may or may not be glycosylated by mammalian or other eukaryotic carbohydrates. In addition, the polypeptides of the invention may comprise initial methionine amino acid residues.

Polypeptides of the invention can be used for a variety of immunomodulatory and inflammatory actions, and can be used for many diseases. Ckβ-8, MIP-4 and Ckβ-1 belong to chemotines and thus they are chemotactic factors for leukocytes (eg monocytes, neutrophils, T lymphocytes, eosinophils, basophils, etc.).

Northern blot analysis revealed that dominantly expressed Ckβ-8, MIP-4 and Ckβ-1 are tissues of hematopoietic cells.

Ckβ-8 has been shown to play an important role in immune response and inflammation. As can be seen in FIG. 22, lipopolysaccharide induces Ckβ-8 from human monocytes. Thus, Ckβ-8 is expressed from monocytes in response to the presence of endotoxins, and Ckβ-8 can be administered to modulate the host's immune response.

As illustrated in FIGS. 10A and 10B, the chemotactic activity of Ckβ-8 against THP-1 cells (A) and PBMC (B) is remarkable. In addition, Ckβ-8 induces significant calcium migration in THP-1 cells (FIG. 11), indicating that Ckβ-8 has a biological effect on monocytes. In addition, Ckβ-8 exhibits dose dependent chemotaxis and calcium migration responses in human monocytes.

Thus, Ckβ-8, MIP-4 and Ckβ-1 can be used to facilitate wound healing by controlling infiltration of target immune cells into the wound site. In a similar manner, the polypeptides of the present invention may enhance host defense against clinical infection, eg, mycobacterial infection by mycobacterial affinity and activation of bactericidal blood cells.

In addition, the polypeptides of the present invention can be used in anti-tumor therapies because there is evidence that chemokine expressing cells injected into the tumor cause a reduction in tumors in the treatment of tumors such as Kaposi's sarcoma. The analysis of FIGS. 12 and 13 illustrates that Ckβ-8 causes THP-1 cells to secrete TNF-α, a known agent that inhibits tumors. Ckβ-8 250 ng / ml induces production and secretion of TNF-α 1200 pg / ml. In addition, Ckβ-8 significantly induces the secretion of other tumor and cancer inhibitors such as IL-6, IL-1 and G-CSF by human monocytes. In addition, Ckβ-8, MIP-4 and Ckβ-1 stimulate the invasion and activation by chemotactic activity of host defense (tumor apoptosis) cells such as cytotoxic T cells and macrophages, and in this way solid Can cure the tumor.

In addition, the polypeptide of the present invention can be used to inhibit proliferation and differentiation of hematopoietic cells, and thus can be used to protect myeloid rod cells from chemotherapeutic agents during chemotherapy. 14 and 15 illustrate that Ckβ-8 and Ckβ-1 inhibit colony formation of hypoproliferative latent colony forming cells, and Ckβ-8 is a potent, specific inhibitor of LPP-CFC colony proliferation. FIG. 16 illustrates that Ckβ-1 specifically inhibits M-CSF stimulated colony formation, but does not inhibit Ckβ-8. However, as can be seen in the figure, both Ckβ-8 and Ckβ-1 specifically inhibit the growth or differentiation of myeloid cells. This antiproliferative effect allows for greater exposure to chemotherapeutic agents and thus more effective chemotherapeutic effects.

The inhibitory effect of Ckβ-1 and Ckβ-8 polypeptides on subpopulations of antigen sensitized progenitor cells (eg granulocytes and macrophages / monocytes) can be used therapeutically to inhibit the proliferation of leukemia cells.

17, 18 and 19, antigen sensitized cells are removed from the cells used and the resulting cell populations are contacted with Ckβ-1 and Ckβ-8. Ckβ-1 caused a decrease in Mac-1 positive cell population by nearly 50%, consistent with the results in FIG. 16 that Ckβ-1 causes inhibition of M-CSF reactive colony-forming cells. As can be seen in FIG. 19, Ckβ-8 inhibits the ability of antigen sensitized progenitor cells to form colonies in response to IL-3, GM-CSF and M-CSF. In addition, as can be seen in Figure 20, the dose response of Ckβ-8 was confirmed to inhibit colony formation. This inhibition may be due to the specific blocking of differentiation signals mediated by these factors or to cytotoxic effects on the progenitor cells.

Polypeptides of the invention can be used, for example, to inhibit T-cell proliferation by inhibiting IL-2 biosynthesis in autoimmune diseases and lymphocytic leukemia.

In addition, Ckβ-8, MIP-4 and Ckβ-1 can be used to inhibit epidermal keratinocyte proliferation (hypercellular hyperplasia) for psoriasis. The reason is that Langerhans cells in the skin have been found to produce MIP-1α.

Ckβ-8, MIP-4 and Ckβ-1 can be used to prevent scars during wound treatment by tissue debridement and replenishment of connective tissue-promoting inflammatory cells and control of excess TGFβ-mediated fibrosis. In addition, these polypeptides can be used for the treatment of seizures, thrombocytopenia, pulmonary embolism and myeloproliferative diseases, because Ckβ-8, MIP-4 and Ckβ-1 increase vascular permeability.

In addition, Ckβ-8 can be used to treat leukemia and abnormally proliferating cells, such as tumor cells, by tricholysis. Ckβ-8 induces apoptosis in a population of hematopoietic progenitor cells as shown in FIG. 21.

Polypeptides of the invention and polynucleotides encoding the polypeptides can be used as research reagents in vitro for scientific research, for synthesis of DNA and for the preparation of DNA vectors, and for the development of therapeutics and diagnostics for the treatment of human diseases. It may be. For example, Ckβ-1 and Ckβ-8 can be used for their expansion by temporarily hindering the differentiation of immature hematopoietic progenitor cells such as granulocytes, macrophages or monocytes. These bone marrow cells can be cultured in vitro.

Fragments of full-length Ckβ-8, MIP-4, or Ckβ-1 genes can be used as hybridization probes for cDNA libraries to isolate the full-length genes and other genes with high sequence similarity or similar biological activity to the genes. Can be separated. However, the probe preferably has at least 30 bases and may contain, for example, at least 50 bases. The probe can also be used to identify cDNA clones corresponding to full-length transcripts and genomic clone (s) containing complete genes including regulatory and promoter regions, exons and introns. One example of the screen includes separating the coding region of the gene by using known DNA sequences to synthesize oligonucleotide probes. Labeled oligonucleotides having sequences complementary to the sequences of genes of the invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine the number of libraries the probe hybridizes to.

The invention also relates to the use of Ckβ-8, MIP-4 and Ckβ-1 as part of a diagnostic assay for detecting disease or susceptibility to the presence of mutations in nucleic acid sequences. Such diseases are associated with subcritical expression of chemokine polypeptides.

Individuals carrying mutations in Ckβ-8, MIP-4 and Ckβ-1 can be detected by various techniques at the DNA level. Diagnostic nucleic acids can be obtained from cells of a patient, such as blood, urine, saliva, tissue biopsy materials and autopsy materials. Genomic DNA can be used directly for detection or enzymatically amplified by PCR prior to analysis (Saiki et al., Nature, 324: 163-166 (1986)). RNA or cDNA can also be used for the same purpose. For example, PCR primers complementary to nucleic acids encoding Ckβ-8, MIP-4 and Ckβ-1 can be used to identify and analyze Ckβ-8, MIP-4 and Ckβ-1 mutations. For example, deletions and insertions can be detected by changes in the size of the amplified product compared to normal genotypes. Point mutations can be identified by hybridizing amplified DNA to radiolabeled Ckβ-8, MIP-4 and Ckβ-1 RNA or alternatively to Ckβ-8, MIP-4 and Ckβ-1 antisense DNA sequences. Perfectly paired sequences can be distinguished from mispaired double sequences by RNase A digestion or melting point differences.

Genetic tests based on DNA sequence differences can be performed by detecting alterations in electrophoretic migration within the gel of DNA fragments in the presence or absence of denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on modified formamide gradient gels where the migration of different DNA fragments is delayed at different locations depending on their specific melting or partial melting points (eg, Myers et al., Science, 230: 1242 (1985). )).

In addition, sequence changes at specific positions can be confirmed by nuclease defense assays or chemical cleavage methods such as RNsae and S1 defenses (eg, Cotton et al., PNAS, USA, 85: 4397-4401 (1985)).

Thus, the detection of specific DNA sequences may include hybridization, RNase defense, chemical cleavage, direct DNA sequencing or methods using restriction enzymes (eg, restriction fragment length polymorphism (RFLP)) and southern blotting of genomic DNA. It can be done by the method.

In addition to the more conventional gel electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

The present invention also relates to diagnostic assays for detecting altered levels of Ckβ-8, MIP-4 and Ckβ-1 proteins in various tissues, for reasons of overexpression of the proteins as compared to normal control tissue samples. This is because the presence of or susceptibility to this disease, for example a tumor, can be detected. Assays used to detect levels of Ckβ-8, MIP-4 and Ckβ-1 proteins in samples derived from the host are known to those of skill in the art, such as radioimmunoassays, competitive-binding assays, Western blot assays. , ELISA analysis, and sandwich analysis. ELISA assays (Coligan et al., Current Protocols in Immunology, 1 (2), Chapter 6 (1991)) initially produce antibodies, preferably monoclonal antibodies, specific for Ckβ-8, MIP-4 and Ckβ-1 antigens. It includes a step. In addition, reporter antibodies are prepared against these monoclonal antibodies. Examples of detectable reagents attached to the reporter antibody include radioactive, fluorescent or horseradish peroxidase enzymes, and in this example horseradish peroxidase enzymes are used. The sample is removed from the host and incubated on a solid support, such as a polystyrene dish, which binds the protein in the sample. Any free protein binding site on the dish is incubated and coated with a nonspecific protein such as BSA. The monoclonal antibody is then incubated in the dish for a time when the monoclonal antibody attaches to any Ckβ-8, MIP-4 and Ckβ-1 protein attached to the polystyrene dish. The reporter antibody bound to horseradish peroxidase is located in the dish and results in binding of the reporter antibody to any monoclonal antibody bound to Ckβ-8, MIP-4 and Ckβ-1. Subsequently, peroxidase substrate is added to the dish, and the amount of color developed over a given time is measured by the amount of Ckβ-8, MIP-4 and Ckβ-1 proteins present in a given patient sample when compared to a standard curve.

Competitive assays can also be used, wherein antibodies specific for Ckβ-8, MIP-4 and Ckβ-1 are attached to the solid support and samples derived from labeled Ckβ-8, MIP-4 and Ckβ-1 and the host The amount of label passing through this support, for example detected by liquid scintillation chromatography, is related to the amount of protein in the sample.

Sandwich analysis is similar to ELISA analysis. In the sandwich assay, Ckβ-8, MIP-4 and Ckβ-1 pass over the solid support and bind to the antibody attached to the solid support. The second antibody is then bound to Ckβ-8, MIP-4 and Ckβ-1. The amount of Ckβ-8, MIP-4 and Ckβ-1 can then be determined by passing a labeled third antibody specific for the second antibody onto the support and binding to the second antibody.

The present invention provides a method for identifying a receptor for a chemokine polypeptide. Genes encoding such receptors can be identified by a variety of methods known to those skilled in the art, for example ligand panning and FACS classification (Coligan et al., Current Protocols in immun. 1 (2), Chapter 5 (1991)). have. Expression cloning can be used, wherein polyadenylated RNA is prepared from cells reactive to the polypeptide and cDNA libraries generated from this RNA are divided into pools and transfected with COS cells and other cells that are not reactive to the polypeptide. Used for Transfected cells grown on glass slides are exposed to labeled polypeptides. The polypeptide can be labeled by a variety of means, including iodide or insertion of a recognition site for a site-specific protein kinase. After immobilization and incubation, the slides are analyzed by automated radiometric analysis. Positive pools are identified, subfuels are prepared, retransfected using an iterative subfouling and rescreening process, and a single clone substantially encoding the putative receptor is obtained.

As another method for receptor identification, the labeled polypeptide can be photoactively bound to the cell membrane, or an article of manufacture expressing the receptor molecule can be extracted. The crosslinked material is dissolved by PAGE analysis and exposed to X-ray film. Labeled complexes containing the receptor of the polypeptide can be excluded, redissolved into peptide fragments, and protein microsequencing can be performed. The amino acid sequences obtained by microsequencing can be used to design a set of degenerate oligonucleotide probes to screen cDNA libraries for identifying genes encoding putative receptors.

The present invention provides methods for screening compounds to identify antagonists and agonists for the chemokine polypeptides of the invention. An agonist is a compound that has a biological function similar to that of the polypeptide, but an antagonist blocks this function. Chemotaxis can be analyzed by placing a cell chewed by any of the polypeptides of the invention on top of a filter having pores of diameter (about 5 μm) sufficient for the cell to permeate. A solution of the potential agent is located at the bottom of the chamber with a suitable control medium in the upper compartment, whereby the concentration gradient of the agent is counted by counting cells that migrate into or through the porous membrane over time. Measure

When assaying for antagonists, chemokine polypeptides of the invention are placed at the bottom of the chamber and potential antagonists are added to determine whether the chemotaxis of the cells is disturbed.

Alternatively, mammalian cell or membrane preparations expressing the receptors of the polypeptide are incubated with radioactivity in the presence of a labeled chemokine polypeptide, eg, the compound. The ability of the compound to block this interaction can then be measured. When assayed for antagonists in this manner, the chemokines are not used and measure the ability of the agonist itself to interact with the receptor.

Examples of potential Ckβ-8, MIP-4 and Ckβ-1 antagonists include antibodies that bind the polypeptide or in some cases oligonucleotides. Other examples of potential antagonists include negative dominant mutants of the polypeptide. Negative dominant mutants are polypeptides that bind to the receptor of a wild type polypeptide but do not retain biological activity.

In addition, antisense constructs are prepared using antisense techniques. Antisense techniques can be used to regulate gene expression via triple helix formation or antisense DNA or RNA, both methods based on binding of polypeptides to DNA or RNA. For example, antisense RNA oligonucleotides of about 10 to 40 base pairs in length can be designed using the 5 'coding region of the polypeptide sequence encoding a mature polypeptide of the invention. DNA oligonucleotides are designed to be complementary to gene regions involved in transcription [triple helix; See Lee et al., Nucl. Acids Res., 6: 3073 (1979); Cooney et al., Science, 241: 456 (1988); And Dervan et al., Science, 251: 1360 (1991)], and can prevent transcription and production of chemokine polypeptides. Antisense RNA oligonucleotides hybridize to mRNA in vivo and block the translation of the mRNA molecule into a polypeptide [antisense; See Okano, J. Neurochem. 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Florida, USA (1988)]. In addition, the oligonucleotides may be delivered to the cell to express the antisense RNA or DNA in vivo to inhibit the production of chemokine polypeptides.

Another potential chemokine antagonist is a peptide derivative of the polypeptide, which is a naturally or synthetically modified analog of the polypeptide, which recognizes and accepts the receptor of the polypeptide, despite losing its biological function. Binding effectively blocks the receptor. Examples of such peptide derivatives include, but are not limited to, small peptides or peptide like molecules.

The antagonist can be used to treat MIP-induced or enhanced diseases such as autoimmune diseases and chronic inflammatory and infectious diseases. Examples of autoimmune diseases include multiple sclerosis and insulin dependent diabetes.

In addition, the antagonist can be used to treat infectious diseases including silicosis, sarcoidosis, idiopathic pulmonary fibrosis by preventing the recruitment and activation of monocytes. They can also be used to treat idiopathic eosinophilia syndrome by preventing eosinophil production and migration. Endotoxin shock can also be treated with the antagonists by preventing migration of macrophages and preventing the production of their chemokine polypeptides of the present invention.

The antagonist may also be used to treat atherosclerosis by preventing monocyte infiltration in the artery walls.

Antagonists can also be used to treat allergic reactions and histological diseases caused by histamine, including late phase allergic reactions, chronic urticaria, atopic dermatitis, which are inhibited by chemokine-induced ductal cells and basophils degranulation and release of histamine. It can also be used to treat allergic reactions caused by IgE, such as allergic asthma, rhinitis, and eczema.

Antagonists can also be used to treat chronic and acute inflammation by preventing the induction of monocytes near the wound. Since chronic and acute inflammatory lung disease is associated with the sequestration of mononuclear phagocytes in the lung, antagonists can be used to regulate macrophages in normal lungs.

Antagonists can be used to treat rheumatoid arthritis by preventing the monocytes from being attracted to the synovial fluid in the joints of the patient. Monocyte influx and activity play an important role in the pathogenesis of degenerative and inflammatory arthrosis.

Antagonists can be used to prevent harmful multisteps primarily due to IL-1 and TNF, which prevent the biosynthesis of other inflammatory cytokines. In this way antagonists can prevent inflammation. Antagonists can be used to inhibit prostaglandin-dependent fever caused by chemokines.

Antagonists can be used to treat cases of bone marrow failure, such as, for example, aplastic anemia and myelodysplastic syndromes.

Antagonists can also be used to treat asthma and allergies by preventing eosinophil accumulation in the lungs. Antagonists can also be used to treat subepithelial basement membrane fibrous tissue proliferation, a salient feature of asthmatic lungs.

The antagonist may be used, for example, in a composition comprising a pharmaceutically acceptable carrier described below.

Chemokine polypeptides and agonists and antagonists can be used in admixture with appropriate pharmaceutical carriers. This composition comprises a therapeutically effective amount of a polypeptide and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should be appropriate for the mode of administration.

The invention also provides a pharmaceutical package or kit comprising one or more containers filled with one or more pharmaceutical composition ingredients of the invention. With regard to the containers, it may be noted that in the form defined by the governmental agency that controls the manufacture, use or sale of pharmaceutical or biological products, which have been approved by the manufacturing, use or sales agency for human administration. In addition, polypeptides and agonists and antagonists can be used with other therapeutic compounds.

The pharmaceutical compositions can be administered by conventional methods such as topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical composition may be administered in an effective amount to treat and / or prevent certain precursors. Generally, polypeptides are administered at least about 10 μg per kg of body weight and in most cases doses of no more than about 8 mg per kg of body weight per day. In most cases, from about 10 μg to about 1 mg per kg of body weight daily, given the route of administration, symptoms, and the like.

Chemokine polypeptides, and agonists or antagonists that are polypeptides, can be used in accordance with the invention to express this polypeptide in vivo, which is often referred to as gene therapy.

Thus, for example, after manipulation of a patient's cells with a polynucleotide (DNA or RNA) that encodes the polypeptide in vitro, the engineered cells can be provided to the patient for treatment with the polypeptide. This method is known in the art. For example, cells can be genetically engineered by methods known in the art using Retrovirus particles comprising RNA encoding a polypeptide of the invention.

Similarly, it may be manipulated in vivo to express the polypeptide in vivo, for example by procedures known in the art. As is known in the art, a production cell that produces a retrovirus particle comprising RNA encoding a polypeptide of the invention is administered to a patient to manipulate the cell in vivo and express the polypeptide in vivo. Such and other methods of administering a polypeptide of the invention by such a method will be apparent to those skilled in the art from the teachings of the invention. For example, expression mediators of genetically engineered cells can be other than retrovirus, such as adenoviruses that can be used in vivo after being combined with appropriate transport mediators.

Retrovirus plasmid vectors include, but are not limited to, Moloney rat sarcoma virus, Moroni rat leukemia virus, spleen necrosis virus, Roth sarcoma virus, and Harvey sarcoma virus.

In a preferred embodiment, the retrovirus expression vector, pMV-7, is laterally linked by the long-term repeats (LTRs) of the Moroni rat sarcoma virus and regulates herpes single virus (HVS) thymidine kinase (tk) promoter. The drug resistance gene neo, which is optional. Unique EcoRI and Hind III sites facilitate insertion of coding sequences (kirschmeier, P. T. et al. DNA, 7: 219-25 (1988))

Vectors include, but are not limited to, Miller et al., Biotechnique vol. No. 9, 980-990 (1989); Retrovirus LTR; SV40 promoter; And one or more suitable promoters, including but not limited to, human cytomegalovirus (CMV) promoters, including but not limited to histones, pol III, and β-actin promoters. Promoters such as eukaryotic cell promoters). The selection of appropriate facilitating factors will be apparent to those skilled in the art from the context of this disclosure.

Nucleic acid sequences encoding polypeptides of the invention include, but are not limited to, viral thymidine kinase promoters such as herpes single thymidine kinase promoter; It is under the control of appropriate promoters, including retroviral LTRs, β-actin promoters, and intrinsic promoters that regulate genes encoding polypeptides.

Retroviral plasmid vectors are used to transduce into packaging cell lines to form producer cell lines. Packaging cells that can be transfected include, but are not limited to, PE501, PA317, and GP + am12. The vector can be transduced into the packaging cells by any method known in the art. This method includes, but is not limited to, electrophoresis, liposome use, and CaPO ₄ precipitation.

Producer cell lines produce infectious retroviral vector particles comprising nucleic acid sequences encoding polypeptides. Retrovirus vector particles can then be used to transduce into eukaryotic cells in vivo or ex vivo. The transfected retrovirus vector can express a nucleic acid sequence encoding a polypeptide. Transduced eukaryotic cells may include, but are not limited to, fibroblasts and endothelial cells.

The sequences of the invention can be used for chromosome identification. In particular, the sequence can target a specific location on each human chromosome and hybridize to that location. In addition, identification of specific sites on the chromosome is currently required. Few chromosome labeling reagents based on actual sequence data (repeated polymorphisms) can be used to indicate chromosomal location. DNA mapping of chromosomes according to the present invention is an important first step that correlates with sequences with genes associated with the disease.

Briefly, sequences can be mapped to chromosomes by making PCR primers (preferably 15-25 bp) from cDNA. Computer analysis of cDNA is used to quickly select primers that do not link one or more exons in genomic DNA, thus making the amplification process difficult. These primers are then used for PCR screening of somatic hybrids containing each human chromosome. Only hybrids comprising human genes corresponding to the primers can produce amplified fragments.

PCR mapping of somatic hybrids is a rapid process of assigning specific DNA to specific chromosomes. Using the present invention in conjunction with the same oligonucleotide primers, sub-locations can be determined through panels of fragments from pools of specific chromosomes or large genomic clones in a similar manner. Other mapping methods that can be mapped to chromosomes in a similar manner include hybridization methods, preliminary screening methods using labeled flow-classified chromosomes, and preselection methods for constructing chromosome specific-cDNA libraries.

Phosphor at the hybridization site of the cDNA clone (FISH) during mid-term chromosome diffusion is used to provide the correct chromosome location in step 1. This technique can be used for short cDNAs such as 500 or 600 bases. For a description of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Technique, Pergamon Press, New York (1988).

Once the sequence is mapped to the correct chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic mapping data. This material is described, for example, in V. McXusick, Mendelian Inheritance in Man (by John Hopkins University Welch Medical Library). Relationships between genes and diseases mapped to the same chromosomal site can be identified through linkage analysis (co-inheritance of physically contiguous genes).

Subsequently, it is necessary to determine the difference in cDNA or genomic sequence between the diseased and uninfected individuals. If mutations are observed in some or all of the diseased but not observed in any normal subject, this mutation is likely to be the cause of the disease.

With current analysis of physical mapping and gene mapping techniques, the exact location of cDNA on chromosomal sites associated with disease is between 50 and 500 potential causal genes (estimated at 1 megabase mapping analysis and 1 gene per 20 kb). .

Polypeptides, fragments or derivatives thereof, or analogs thereof, or cells expressing the same can be used as immunogens to generate antibodies to them. These antibodies can be, for example, polyclonal or monoclonal antibodies. The invention also encompasses antibodies that can be applied to chimeric, single chain and human as well as Fab fragments or products of Fab expression libraries. Various procedures known in the art can be used for the production of antibodies and fragments.

Antibodies produced against a polypeptide corresponding to a sequence of the invention or its receptor in vivo can be obtained by injecting the polypeptide directly into an animal or by administering the polypeptide to an animal, preferably a human body. The antibody thus obtained binds to the polypeptide. In this method, only sequences encoding polypeptide fragments can be used to generate antibodies that bind the entire native polypeptide. The antibody is then used to separate the polypeptide from tissue expressing the polypeptide.

To prepare monoclonal antibodies, any technique that provides antibodies produced by continuous cell line culture may be used. Examples include hybridoma technology (Kohler and Milstein, 1975, Nature, 256: 495-497), trioma technology, human B-cell hybridoma technology (Kozbor et al., 1983, Immunology Today 4: 72), and human body single. EBV-Hybridoma technology (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R, Liss, Inc, p 77-96) for producing antibodies.

Techniques for the production of single chain antibodies (US Pat. No. 4,946,778) can be applied to generate single chain antibodies against the polypeptide products of the immunogens of the invention. In addition, the transgenic mice can be used to express antibodies applicable to the human body to the immunogenic polypeptide product of the present invention.

Further, the present invention will be described with reference to the following examples, but it should be appreciated that the present invention is not intended to be limited to these examples. All parts or quantities are by weight unless otherwise indicated.

Some frequently occurring methods and / or terms will be described in order to facilitate understanding of the following examples.

Plasmids are indicated by a lowercase p before and / or after an uppercase and / or number. The starting plasmids herein may be structured from commercially available, publicly available non-limiting bases, or available plasmids according to published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to those of ordinary skill in the art.

Degradation of DNA refers to catalytic cleavage of DNA with restriction enzymes that act on specific sites in the DNA. Various restriction enzymes used herein are commercially available and use reaction conditions, cofactors and other requirements known to those skilled in the art. For analysis, 1 μg of plasmid or DNA fragment is typically used with about 2 units of enzyme in about 20 μl buffer. To separate DNA fragments for plasmid structure, typically 5-50 μg DNA is digested with 20-250 enzyme units at larger doses. Suitable buffers and substrate amounts of certain restriction enzymes are determined by the manufacturer. An incubation time of about 1 hour at 37 ° C. is used but may vary as directed by the supplier. After digestion, the reaction is electrophoresed directly onto the polyamide gel to separate the desired fragments.

Separation by size of fragmented fragments is described by Goeddel, D. et al. Nucleic Acids Res. 8: 4057 (1980), using an 8% polyamide gel.

Oligonucleotides refer to a single chain polydeoxynucleotide or two complementary polydeoxynucleotide chains that can be chemically synthesized. Synthetic oligonucleotides are free of 5 'phosphate and thus cannot be linked to oligonucleotides without adding phosphate using ATP in the presence of kinases. Synthetic oligonucleotides are linked to fragments that are not dephosphorylated.

Linking refers to the process of forming phosphodiester between two-stranded nucleic acid fragments (Maniatis, T. et al., Id., P 146). Without other suggestions, ligation can be performed using known buffers and 10 units of T4 DNA ligase (ligase) per 0.5 μg of linked DNA fragments of approximately the same mole.

Unless otherwise noted, transformations were found in Graham, F. and Van der Eb. A., carried out by the method described in Virology's method.

Polypeptide Ckβ-1 of the present invention named MIP-1γ in the parent application herein is a novel component of β chemokines based on amino acid sequence homology. Ckβ-8 polypeptides, originally called MIP-3 in the parent application, are also novel components of β chemokines, based on amino acid sequence homology.

According to a first aspect of the present invention there is provided novel mature polypeptides of human Ckβ-8, human MIP-4 and human Ckβ-1 and their biologically active, diagnostic or therapeutically useful fragments, analogs and derivatives thereof.

According to a second aspect of the invention there is provided an isolated nucleic acid molecule encoding said polypeptide, including mRNA, DNA, cDNA, genomic DNA, as well as biologically active and diagnostic or therapeutically useful fragments, analogs and derivatives thereof. .

According to a third aspect of the present invention, a recombinant technique comprising culturing recombinant prokaryotic host cells and / or eukaryotic host cells containing a nucleic acid sequence, wherein said polypeptide is subjected to conditions that enhance expression of said protein and then said protein recovery. Provide a way to produce.

According to a fourth aspect of the invention, the polypeptide or polynucleotide encoding the polypeptide is protected for bone marrow hepatic cells from chemotherapy during treatment, such as chemotherapy, eliminates leukemia cells, and stimulates an immune response. And modulate hematopoiesis and lymphocyte attraction, treat psoriasis and tumor bodies, improve host defense against resistant chronic and acute infections, and stimulate wound healing.

According to a fifth aspect of the invention, an antibody against said polypeptide is provided.

According to a sixth aspect of the present invention, the inhibitory action of the polypeptide can be used, for example, to inhibit the production of IL-1 and TNF-α and to treat aplastic anemia, myelodysplastic syndrome, asthma and arthritis. Provides an antagonist to the polypeptide.

According to a seventh aspect of the invention, the invention also provides nucleic acid probes containing nucleic acid molecules of sufficient length to specifically hybridize to Ckβ-8, Ckβ-1 and MIP-4 nucleic acid sequences.

According to an eighth aspect of the invention, the invention provides diagnostic assays for detecting diseases associated with low and overexpression of polypeptides and for detecting mutations in nucleic acid sequences encoding such polypeptides.

According to a ninth aspect of the invention, the invention provides a therapeutic agent and diagnostic agent for treating said polypeptide or polynucleotide encoding said polypeptide for in vitro testing relating to scientific research, DNA synthesis and DNA vector production, and for treating human diseases. Provided are methods for use as research reagents for development.

These and other aspects of the invention will be apparent to those skilled in the art from the teachings herein.

Example 1

Bacterial Expression and Purification of Ckβ-8

DNA sequences encoding Ckβ-8, ATCC # 75676 were initially prepared using PCR oligonucleotide primers corresponding to the 5 'and 3' terminal sequences (excluding signal peptide sequences) and vector sequence 3 'of the processed Ckβ-8 protein. Is amplified by Ckβ-8. In addition, nucleotides corresponding to Bam HI and XbaI are added to the 5 'and 3' sequences, respectively. The 5 'oligonucleotide primer having the 5' TCAGGATCCGTCACAAAAGATGCAGA 3 '(SEQ ID NO: 7) comprises 18 nucleotides of the Ckβ-8 coding sequence starting from the amino acid presumed to be the end of the processed protein, followed by the BamHT restriction enzyme site. 3 'SEQ ID NO: 5' CGCTCATAGAGTAAAACGACGGCCAGT 3 '(SEQ ID NO: 8) comprises the complementary sequence for the XbaI site. This restriction enzyme site corresponds to the restriction enzyme site on the bacterial expression vector pQE-9 (Qiagen Inc.). pQE-9 encodes antibiotic resistance (Amp ^I ), bacterial origin of replication (ori), IPTG-regulated facilitation effector (P / O), ribosomal binding site (RBS), 6-His tag and restriction enzyme site. PQE-9 is then degraded to BamHI and XbaI. The amplified sequence is bound into pQE-9 and inserts into the frame the sequence encoding the histidine tag and RBS. The binding mixture is then used to transform into E. coli strain M15 / rep4 available from Quinagogen Inc. M15 / rep4 contains multiple copies of plasmid pREP4, which expresses lacI inhibitory factor and confers kanamycin resistance (Kan ^I ). Transformants are identified by their ability to proliferate on LB plates and ampicillin / kanamycin resistant colonies are selected. Plasmid DNA was isolated and restriction analysis performed. Colons containing the desired structure were grown overnight (O / N) in liquid culture in LB medium supplemented with Amp (100 ug / ml) and Kan (25 ug / ml). O / N culture was used to inoculate the large culture at a ratio of 1: 100 to 1: 250. Cells were grown at an optical density of 600 (OD ⁶⁰⁰ ) of 0.4 to 0.6. IPTG (isopropyl-BD-thiocalacto pyranoside) was added at a final concentration of 1 mM. IPTG was induced by inactivating lacI inhibitors that eliminate P / O associated with increased gene expression. Cells were expanded for an additional 3-4 hours. Cells were then obtained by centrifugation. Cell pellets were solubilized in 6 moles of guotropic guanidine HCl. After fractionation, solubilized Ckβ-8 was purified from solution by chromatography on a nickel-chelate column under conditions that strongly bound the protein containing the 6-His tag (Hochuli, E et al., J. Chromatography 411: 177-184). (1984)). Ckβ-8 (95% purity) is eluted from the column under 6 molar guanidine HCl pH 5.0 and adjusted for recovery with 3 molar guanidine HCl, 100 mM sodium phosphate, 10 mM glutathione (oxidation) and 2 mM glutathione (oxidation). . After incubating the solution for 12 hours, the protein is dialyzed with 10 mM sodium phosphate.

Example 2

Bacterial Expression and Purification of MIP-4

DNA sequences encoding MIP-4, ATCC # 75675 were initially amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences (excluding signal peptide sequences) of the processed MIP-4 protein. In addition, nucleotides corresponding to Bam HI and XbaI were added at the 5 'and 3' ends, respectively. The 5 'oligonucleotide primer having the sequence 5' TCAGGATCCTGTGCACAAGTTGGTACC 3 '(SEQ ID NO: 9) comprises 18 nucleotides of the MIP-4 coding sequence starting from the amino acid presumed to be the end of the processed protein codon following the BamHI restriction enzyme site; 3 'SEQ ID NO: 5' CGCTCTAGAGTAAAACGACGGCCAGT 3 '(SEQ ID NO: 10) comprises the complementary sequence for the XbaI site. This restriction enzyme site corresponds to the restriction enzyme site on the bacterial expression vector pQE-9 (Qiagen Inc., located in Chatsworth, California). pQE-9 encodes antibiotic resistance (Amp ^I ), bacterial origin of replication (ori), IPTG-regulated promoter effector (P / O), ribosomal binding site (RBS), 6-His tag, and restriction enzyme site . PQE-9 is then digested with BamHI and XbaI and the amplified sequence is incorporated into pQE-9 and inserts into the frame the sequence encoding the histidine tag and RBS. The binding mixture is then used to transform into E. coli strain M15 / rep4 available from Quinagogen Inc. M15 / rep4 contains multiple copies of plasmid pREP4, which expresses lacI inhibitory factor and confers kanamycin resistance (Kan ^I ). Transformants are identified by their ability to proliferate on LB plates and ampicillin / kanamycin resistant colonies are selected. Plasmid DNA was isolated and restriction analysis performed. Colons containing the desired structure were grown overnight (O / N) in liquid culture in LB medium supplemented with Amp (100 ug / ml) and Kan (25 ug / ml). O / N culture was used to inoculate the large culture at a ratio of 1: 100 to 1: 250. Cells were grown at an optical density of 600 (OD ⁶⁰⁰ ) of 0.4 to 0.6. IPTG (isopropyl-BD-thiocalacto pyranoside) was added at a final concentration of 1 mM. IPTG was induced by inactivating lacI inhibitors that eliminate P / O associated with increased gene expression. Cells were expanded for an additional 3-4 hours. Cells were then obtained by centrifugation. Cell pellets were solubilized in 6 moles of guotropic guanidine HCl. After fractionation, the solubilized MIP-4 was purified from solution by chromatography on a nickel-chelate column under conditions that strongly bound the protein containing the 6-His tag (Hochuli, E et al., J. Chromatography 411: 177-184). (1984)). MIP-4 (95% purity) is eluted from the column under 6 mol guanidine HCl pH 5.0 and adjusted for recovery with 3 mol guanidine HCl, 100 mM sodium phosphate, 10 mM glutathione (oxidation) and 2 mM glutathione (oxidation). . After incubating the solution for 12 hours, the protein is dialyzed with 10 mM sodium phosphate.

Example 3

Bacterial Expression and Purification of Ckβ-1

DNA sequences encoding Ckβ-1, ATCC # 75572 were initially amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences (excluding signal peptide sequences) of the processed Ckβ-1 protein, and further Nucleotides corresponding to Bam HI and XbaI were added at the 5 'and 3' ends, respectively. The 5 'oligonucleotide primer having the sequence 5' GCCCGCGGATCCTCCTCACGGGGACCTTAC 3 '(SEQ ID NO: 11) comprises 15 nucleotides of the Ckβ-1 coding sequence starting from the amino acid presumed to be the end of the processed protein codon following the BamHI restriction enzyme site; 3 'SEQ ID NO: 5' GCCTGCTCTAGATCAAAGCAGGGAAGCTCCAG 3 '(SEQ ID NO: 12) comprises the last 20 nucleotides of the complementary sequence, the translation stop codon, and the Ckβ-1 coding sequence for the XbaI site. This restriction enzyme site corresponds to the restriction enzyme site on the bacterial expression vector pQE-9 (Qiagen Inc., located in Chatsworth, California). pQE-9 encodes antibiotic resistance (Amp ^I ), bacterial origin of replication (ori), IPTG-regulated facilitation effector (P / O), ribosomal binding site (RBS), 6-His tag and restriction enzyme site . PQE-9 is then digested with BamHI and XbaI and the amplified sequence is incorporated into pQE-9 and inserts into the frame the sequence encoding the histidine tag and RBS. The binding mixture is then obtained from E. co. Used to transform into coli strain M15 / rep4. M15 / rep4 contains multiple copies of plasmid pREP4, which expresses lacI inhibitory factor and confers kanamycin resistance (Kan ^I ). Transformants are identified by their ability to proliferate on LB plates and ampicillin / kanamycin resistant colonies are selected. Plasmid DNA was isolated and restriction analysis performed. Colons containing the desired structure were grown overnight (O / N) in liquid culture in LB medium supplemented with Amp (100 ug / ml) and Kan (25 ug / ml). O / N culture was used to inoculate the large culture at a ratio of 1: 100 to 1: 250. Cells were grown at an optical density of 600 (OD ⁶⁰⁰ ) of 0.4 to 0.6. IPTG (isopropyl-BD-thiocalacto pyranoside) was added at a final concentration of 1 mM. IPTG was induced by inactivating lacI inhibitors that eliminate P / O associated with increased gene expression. Cells were expanded for an additional 3-4 hours. Cells were then obtained by centrifugation. Cell pellets were solubilized in 6 moles of guotropic guanidine HCl. After fractionation, the solubilized MIP-4 was purified from solution by chromatography on a nickel-chelate column under conditions that strongly bound the protein containing the 6-His tag (Hochuli, E et al., J. Chromatography 411: 177-184). (1984)). Ckβ-1 (95% purity) is eluted from the column under 6 molar guanidine HCl pH 5.0 and adjusted for recovery with 3 molar guanidine HCl, 100 mM sodium phosphate, 10 mM glutathione (oxidation) and 2 mM glutathione (oxidation). . After incubating the solution for 12 hours, the protein is dialyzed with 10 mM sodium phosphate.

Example 4

Expression of Recombinant Ckβ-8 in COS Cells

Expression of the plasmid CMV-Ckβ-8 HA is shown by 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E. Coli replication origin, 4) vector pcDNAI / Amp (extracellular production) comprising CMV promoters following multiple binding sites, SV40 introns and polyadenylation sites. Recombinant protein expression is regulated by CMV promoters because the fragment encoding the HA tag fused in-frame to the complete Ckβ-8 precursor and 3 'end is cloned into multiple bound regions of the vector. HA tags corresponding to epitopes were derived from influenza hemagglutinin protein as described above (I. Wilson et al., Cell, 37: 767 (1984)). Injecting an HA tag into a target protein facilitates detection of a recombinant protein with an antibody recognized by the HA epitope.

The plasmid structure method is as follows:

DNA sequence encoding Ckβ-8, ATCC # 75676 is structured by PCR using two primers; 5 ′ primer 5 ′ GGAAAGCTTATGAAGGTCTCCGTGGCT 3 ′ (SEQ ID NO: 13) comprises 18 nucleotides of the Ckβ-8 coding sequence starting from the initiation codon after the HindIII site; 3 'SEQ ID NO: 5' CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAA TTCTTCCTGGTCTTGATCC 3 '(SEQ ID NO: 14) comprises the final 20 nucleotides (not including stop codon) of the complementary sequence, translation stop codon, HA tag, and Ckβ-1 coding sequence for the XbaI site. Thus, the PCR product comprises a HindIII site, a Ckβ-8 sequence leading to a HA tag fused in frame, a translation termination stop codon adjacent to the HA tag, and an XbaI site. PCR amplified DNA fragments and vectors, pcDNAI / Amp, are digested and linked by HindIII and XbaI restriction enzymes. This coupling mixture. Coli strain SURE (Stratagen Cloning Systems, La Giulia, Calif.) Is transformed, the transformed culture is plated on an ampicillin medium plate, and resistant colonies are selected. Plasmid DNA is isolated from the transformants and tested in restriction assays for the presence of correct fragments. For expression of recombinant Ckβ-8, COS cells were transfected with expression vectors by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning; A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Infect. Expression of Ckβ-8-HA protein is detected using radiolabeling and immunoprecipitation methods (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Two days after transfection, cells are labeled with ³⁵ S-cysteine for 8 hours. The cultures are harvested and the cells lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.1% DOC, 50 mM Tris, pH 7.5) (Wilson, I et al., Id 37: 767 (1984)) Cell lysates and cell cultures are precipitated with HA specific monoclonal antibodies The precipitated proteins are analyzed on a 15% SDS-PAGE gel.

Example 5

Expression of Recombinant MIP-4 in COS Cells

Expression of the plasmid CMV-MIP-4 HA is shown by 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E. Coli replication origin, 4) vector pcDNAI / Amp (extracellular production) comprising CMV promoters following multiple binding sites, SV40 introns and polyadenylation sites. Recombinant protein expression is regulated by CMV promoters because the fragment encoding the HA tag fused in-frame to the complete Ckβ-8 precursor and 3 'end is cloned into multiple bound regions of the vector. HA tags corresponding to epitopes were derived from influenza hemagglutinin protein as described above (I. Wilson et al., Cell, 37: 767 (1984)). Injecting an HA tag into a target protein facilitates detection of a recombinant protein with an antibody recognized by the HA epitope.

The plasmid structure method is as follows:

DNA sequences encoding MIP-4, ATCC # 75675, are structured by PCR using two primers; 5 'primer 5' GGAAAGCTTATGAAGGGCCTTGCAGCTGCC 3 '(SEQ ID NO: 15) comprises 20 nucleotides of the MIP-4 coding sequence starting from the start codon after the HindIII site; 3 'SEQ ID NO: 5' CGCTCTAGATCAABCGTAGTCTGGGACGTCGTATGGGTAG GCATTCAGCTTCAGGTC 3 '(SEQ ID NO: 16) comprises the last 19 nucleotides (not including stop codon) of the complementary sequence, translation stop codon, HA tag, and MIP-4 coding sequence for the XbaI site. Thus, the PCR product comprises a HindIII site, a MIP-4 sequence leading to a HA tag fused in frame, a translation termination stop codon adjacent to the HA tag, and an XbaI site. PCR amplified DNA fragments and vectors, pcDNAI / Amp, are digested and linked by HindIII and XbaI restriction enzymes. This coupling mixture. Coli strain SURE (Stretagen Cloning Systems, La Giulia, Calif.) Is transformed, the transformed culture is plated on an ampicillin medium plate, and resistant colonies are selected. Plasmid DNA is isolated from the transformants and tested in restriction assays for the presence of correct fragments. For expression of recombinant MIP-4, COS cells were transfected with expression vectors by DEAE-DEXTRAN methods (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning; A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Infect. Expression of MIP-4-HA protein is detected using radiolabeling and immunoprecipitation methods (E. Harlow, D. Lane, Antibodlies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Two days after transfection, cells are labeled with ³⁵ S-cysteine for 8 hours. The cultures are harvested and the cells lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.1% DOC, 50 mM Tris, pH 7.5) (Wilson, I et al., Id 37: 767 (1984)) Cell lysates and cell cultures are precipitated with HA specific monoclonal antibodies The precipitated proteins are analyzed on a 15% SDS-PAGE gel.

Example 6

Expression of Recombinant Ckβ-1 in COS Cells

The expression of CMV-Ckβ-1 HA was 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E. Coli replication origin, 4) vector pcDNAI / Amp (extracellular production) comprising CMV promoters following multiple binding sites, SV40 introns and polyadenylation sites. Recombinant protein expression is regulated by CMV promoters because the fragment encoding the HA tag fused in-frame to the complete Ckβ-1 precursor and 3 'end is cloned into multiple bound regions of the vector. HA tags corresponding to epitopes were derived from influenza hemagglutinin protein as described above (I. Wilson et al., Cell, 37: 767 (1984)). Injecting an HA tag into a target protein facilitates detection of a recombinant protein with an antibody recognized by the HA epitope.

The plasmid structure method is as follows:

The DNA sequence encoding Ckβ-1, ATCC # 75572 is structured by PCR using two primers; 5 ′ primer 5 ′ GGAAAGCTTATGAAGATTCCGTGGCTGC 3 ′ (SEQ ID NO: 17) comprises 20 nucleotides of the Ckβ-1 coding sequence starting from the start codon after the HindIII site; 3 ′ SEQ ID NO: 5 ′ CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAG TTCTCCTTCATGTCCTTG 3 ′ (SEQ ID NO: 18) comprises the final 19 nucleotides (not including stop codon) of the complementary sequence, stop translation codon, HA tag, and Ckβ-1 coding sequence for the XbaI site. Thus, the PCR product comprises a HindIII site, a Ckβ-1 sequence leading to a HA tag fused in frame, a translation termination stop codon adjacent to the HA tag, and an XbaI site. PCR amplified DNA fragments and vectors, pcDNAI / Amp, are digested and linked by HindIII and XbaI restriction enzymes. This coupling mixture. Coli strain SURE (Stretagen Cloning Systems, La Giulia, Calif.) Is transformed, the transformed culture is plated on an ampicillin medium plate, and resistant colonies are selected. Plasmid DNA is isolated from the transformants and tested in restriction assays for the presence of correct fragments. For expression of recombinant Ckβ-1, COS cells were transfected with expression vectors by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning; A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Infect. Expression of Ckβ-1-HA protein is detected using radiolabeling and immunoprecipitation methods (E. Harlow, D. Lane, Antibodlies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Two days after transfection, cells are labeled with ³⁵ S-cysteine for 8 hours. The cultures are harvested and the cells lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.1% DOC, 50 mM Tris, pH 7.5) (Wilson, I et al., Id 37: 767 (1984)) Cell lysates and cell cultures are precipitated with HA specific monoclonal antibodies The precipitated proteins are analyzed on a 15% SDS-PAGE gel.

Example 7

Expression Patterns of Ckβ-8 in Human Tissue

Northern blot analysis was performed to test the expression level of Ckβ-8 in human tissue. RNA samples from total cells were isolated by RNAzol ^™ B system (Biotex Laboratories Inc., Houston, 77033, Texas). About 10 ug of total RNA isolated from each specific human tissue is isolated on a 1% agar gel and blotted with nylon filters (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Press, (1989)). Labeling reactions were performed according to the Stratagen Prime-It kit with 50ng DNA fragments. Labeled DNA is purified on a Select-G-50 column (5 Prime-3 Prime, Inc. Boulder, CO). The filter is then hybridized overnight at 65 ° C. with 1,000,000 cpm / ml radiolabeled total length Ckβ-8 gene in 0.5M NaPO ₄ , pH 7.4 and 7% SDS. After washing twice at room temperature and twice at 60 ° C. with 0.5 × SSC, 0.1% SDS, the filter is exposed overnight at −70 ° C. using a strengthening screen.

Example 8

Expression Patterns of MIP-4 in Human Tissue

Northern blot analysis was performed to test the expression level of MIP-4 in human tissue. RNA samples from total cells were isolated by RNAzol ^™ B system (Biotex Laboratories Inc., Houston, Texas). About 10 ug of total RNA isolated from each specific human tissue is isolated on a 1% agar gel and blotted with nylon filters (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Press, (1989)). Labeling reactions were performed according to the Stratagen Prime-It kit with 50ng DNA fragments. Labeled DNA is purified on a Select-G-50 column (5 Prime-3 Prime, Inc. Boulder, CO). The filter is then hybridized overnight at 65 ° C. with 1,000,000 cpm / ml radiolabeled total length MIP-4 gene in 0.5M NaPO ₄ , pH 7.4 and 7% SDS. After washing twice at room temperature and twice at 60 ° C. with 0.5 × SSC, 0.1% SDS, the filter is exposed overnight at −70 ° C. using a strengthening screen.

Example 9

Expression Pattern of Ckβ-1 in Human Tissue

Northern blot analysis was performed to test the expression level of Ckβ-1 in human tissue. RNA samples from total cells were isolated by RNAzol ^™ B system (Biotex Laboratories Inc., Houston, Texas). About 10 ug of total RNA isolated from each specific human tissue is isolated on a 1% agar gel and blotted with nylon filters (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Press, (1989)). Labeling reactions were performed according to the Stratagene Prime-It kit with 50ng DNA fragments. Labeled DNA is purified on a Select-G-50 column (5 Prime-3 Prime, Inc. Boulder, CO). The filter is then hybridized overnight at 65 ° C. with 1,000,000 cpm / ml radiolabeled total length Ckβ-1 gene in 0.5 NaPO ₄ , pH 7.4 and 7% SDS. After washing twice at room temperature and twice at 60 ° C. with 0.5 × SSC, 0.1% SDS, the filter is exposed overnight at −70 ° C. using a strengthening screen.

Example 10

Expression and Purification of Chemokine Ckβ-8 Using Baculovirus Expression System

SF9 cells were infected with recombinant baculovirus to express Ckβ-8 cDNA. Cells were infected at 2 MOI and incubated at 28 ° C. for 72-96 hours. Cell debris was removed from the infected culture by a low speed centrifuge. Protease inhibitor cocktails were added to the supernatant at a final concentration of 20 μg / ml Pefabloc SC, 1 μg / ml leupeptin, 1 μg / ml E-64 and 1 mM EDTA. The concentration of Ckβ-8 in the supernatant was monitored by loading 20-30 μl of the supernatant on a 15% SDS-PAGE gel. Ckβ-8 was detected as a visible 9Kd band corresponding to several mg of expression concentration per liter. Further Ckβ-8 was purified by a three step purification procedure: heparin binding affinity chromatography. The supernatant of Baculovirus culture was mixed with 1/3 volume of buffer containing 100 mM HEPES / MES / NaOAc pH 6 and filtered through a 0.22 μm membrane. The sample was then applied to a heparin binding column (HE1 Porous 20, Bio-Perceptive System Incorporated). Ckβ-08 was eluted at about 300 mM NaCl in a linear gradient of 50-500 mM NaCl in 50 mM HEPES / MES / NaOAc at pH 6; Cation exchange chromatography. Ckβ-8, concentrated from heparin chromatography, is diluted 5-fold with a buffer containing 50 mM HEPES / MES / NaOAc at pH 6. The resulting mixture was applied to a cation exchange column (S / M Porous 20, Bio-Perceptive System Incorporated). Ckβ-8 was eluted at about 250 mM NaCl in a 25-300 mM NaCl linear gradient in 50 mM HEPES / MES / NaOAc at pH 6; Size exclusion chromatography. Following cation exchange chromatography, Ckβ-8 was further purified by application to a size exclusion column (HW50, TOSO HAAS, 1.4 × 45 cm). Ckβ-8 was fractionated at a position close to the 13.7 Kd molecular weight criterion (RNase A) corresponding to the dimeric form of the protein.

After this three step purification, the resulting Ckβ-8 was determined to have greater than 90% purity from Coomassie Blue staining of the SDS-PAGE gel (FIG. 9).

Purified Ckβ-8 was also tested for endotoxin / LPS impurities. LPS content was less than 0.1 ng / ml according to LAL analysis (BioWhittaker).

Example 11

Effect of Baculovirus-expressed Ckβ-1 and Ckβ-8 on M-CSF and SCF-stimulated Colony Formation of Newly Isolated Myeloid Cells

Low density populations of rat bone marrow cells were incubated in treated tissue culture dishes at 37 ° C. for 1 hour to remove monocytes, macrophages, and other cells that adhere to the plastic surface. Non-adherent populations of cells were plated (10,000 cells / dish) in agar containing growth medium with or without the factors shown in FIG. 16. Cultures were incubated at 37 ° C. for 10 days (88% N ₂ , 5% CO ₂ , and 7% O ₂ ) and colonies were counted under reverse microscope. Data were presented as mean colony counts and obtained from three analyzes.

Example 12

Effects of Ckβ-8 and Ckβ-1 on IL-3 and SCF-stimulated Proliferation and Differentiation of Myeloid Cell Homologous Populations

Populations of murine bone marrow cells enriched in progenitor cells of progenitors were obtained using a negative selection method, with the majority of the series of related cells being a panel of monoclonal antibodies (anti-cd11b, CD4, CD8, CD45R, and Gr-1 antigens). And magnetic beads. The resulting populations (cognate cells) were plated in the presence or absence of the indicated chemokines (50 ng / ml) in growth medium supplemented with IL-3 (5 ng / ml) and SCF (100 ng / ml). After 7 days incubation at 37 ° C. in a humidified thermostat (5% CO ₂ , 7% O ₂ and 88% N ₂ conditions), cells were obtained and analyzed for HPP-CFCs and immature progenitor cells. In addition, cells were analyzed for expression of specific differentiated antigens by FACScan. Colony data are expressed as mean colony number +/- standard deviation and data were obtained from analyzes performed in six dishes for each population of cells (FIG. 17).

Example 13

Inhibition of Ckβ-8 on colony formation in response to IL-3, M-CSF and GM-CSF

Rat bone marrow cells were perfused in the femur and tibia, separated into picol density gradients and monocytes removed by plastic attachment. The resulting population of cells overnight in MEK-based medium supplemented with IL-3 (5 ng / ml), GM-CSF (5 ng / ml), M-CSF (10 ng / ml) or G-SCF (10 ng / ml) Incubated. These cells were subjected to agar colony formation analysis under IL-3 (5ng / ml), GM-CSF (5ng / ml) or M-CSF (5ng / ml) with or without 50 ng / ml Ckβ-8. Plates were plated at 1,000 cells / plate. Data show colony formation as a percentage of the number of colonies formed by each specific factor. Two experiments are shown as the mean value of two replicate culture dishes with error bars representing the standard deviation of each experiment (FIG. 19).

Example 14

Expression by Gene Therapy

Fibroblasts were obtained from specimens by skin biopsy. The resulting tissue was placed in tissue culture medium and separated into bovine fragments. A small mass of tissue was placed on the wet surface of the tissue culture flask and about 10 fragments were placed in each flask. The flask was inverted, tightly sealed and kept at room temperature overnight. After 24 hours at room temperature, the flask was inverted and fresh medium (eg, F12 medium of ham with 10% FBS, penicillin and streptomycin) was added with the tissue mass fixed at the bottom of the flask. Then it was incubated for about 1 week at 37 ℃. At this point, fresh medium was added and then changed once every 7 days. After incubation for a further 2 weeks, a monolayer consisting of fibroblasts appeared. The monolayer was trypsinized and expanded to larger flasks.

PMV-7 (Kirschmeier, P.T. et al., DNA 7: 219-25 (1988)) adjacent to the long terminal repeats of the Moroni rat sarcoma virus were digested with EcoRI and HindIII and then treated with calf pancreatic phosphatase. The linear vector was fractionated on agar gel and purified using glass beads.

CDNAs encoding polypeptides of the invention were amplified using PCR primers corresponding to 5 'and 3' sequences, respectively. The 5 'primer contained the EcoRI position and the 3' primer contained the HindIII position. Equal amounts of Moroni rat sarcoma virus linear backbone and EcoRI and HindIII fragments were added together in the presence of T4 DNA ligase. The resulting mixture was maintained under suitable conditions for linking the two fragments. Bacterial HB101 was transformed using the ligation mixture and then plated on agar containing kanamycin to confirm that the desired gene was properly inserted into the vector.

Ampotropic pA317 or GP + am12 packaging cells were grown to confluence density by Dulbecco's modified Eagle's medium cold tissue culture with 10% calf serum (CS), penicillin and streptomycin. MSV vectors containing the genes were then added to the medium, and the packaging cells were transformed with the vectors. The packaging cells produced infectious virus particles containing the gene (hereinafter, the packaging cells are referred to as manufacturer cells).

Fresh medium is added to the transduced production cells, and then the medium is obtained from 10 cm plates of crowded producer cells. Scattering medium containing infectious virus particles is used to filter adherent cells to remove adherent producer cells and infect the medium with fibroblasts. The medium is removed from the subpopulation plate of the fibroblasts and the medium is quickly replaced with producer cells. Remove this medium and replace with fresh medium. If the virus titer is high, eventually all fibroblasts are infected and do not need to be selected. If the titer is very low, you will need to use a retrovirus vector to select markers such as neo or his .

Engineered fibroblasts are injected either alone or in a multiplyed host to colonize on cytodex 3 microcarrier beads. Fibroblasts can then produce protein products.

Various modifications and variations of the present invention are possible in light of the above teachings, and thus the present invention may be practiced in the appended claims in addition to those specifically described.

Standing column list

(1) General Information:

(i) Applicant:

(A) Name: Lee, etc.

(ii) Name of the invention: human chemokine beta-8, chemokine beta-1 and macrophage inflammatory protein-4

(iii) Number of sequences: 18

(iv) Letter Address:

(A) Recipients: Kaela, Byrne, Bain, Gilphyllan, Cinch, Steward and Allstein

(B) Street: Becker Farm Road 6

(C) City: Roseland

(D) State: New Jersey

(E) Country: United States

(F) ZIP: 07068

(v) computer readable form:

(A) Media Type: 3.5 Inch Diskette

(B) Computer: IBM PS / 2

(C) operating system: MS-DOS

(D) Software: WordPerfect 5.1

(vi) Prior application data:

(A) Application number:

(B) filing date:

(C) Classification code:

(vii) prior application data:

(A) Application Number: US 08 / 173,209

(B) Application date: December 22, 1993

(viii) prior application data:

(A) Application number: US 08 / 208,339

B) Application Date: March 8, 1994

(ix) prior application data:

(A) Application number: PCT / US94 / 07256

(B) Application date: June 28, 1994

(ix) Agent Information:

(A) Statement: Ferraro, Gregory D.

(B) Registration Number: 36,134

(C) Reference Number: 325800-289

(x) communication information:

(A) Telephone: 201-994-1700

(B) Fax: 201-994-1744

(2) Information about SEQ ID NO: 1

(i) Sequence Properties:

(A) Length: 363 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

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(xi) SEQ ID NO: SEQ ID NO: 1:

(2) Information about SEQ ID NO:

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(D) Form: straight chain

(ii) Molecular form: protein

(xi) SEQ ID NO: SEQ ID NO: 2:

(2) Information about SEQ ID NO:

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(A) Length: 282 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

(ii) Molecular form: cDNA

(xi) SEQ ID NO: SEQ ID NO: 3:

(2) Information about SEQ ID NO:

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(xi) SEQ ID NO: SEQ ID NO: 4:

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(xi) SEQ ID NO: SEQ ID NO: 16:

(2) Information about SEQ ID NO: 17:

(i) Sequence Properties:

(A) Length: 28 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

(ii) Molecular form: oligonucleotide

(xi) SEQ ID NO: SEQ ID NO: 17:

(2) Information about SEQ ID NO: 18:

(i) Sequence Properties:

(A) Length: 58 base pairs

(B) Sequence type: nucleic acid

(C) Number of chains: 1 chain

(D) Form: straight chain

(ii) Molecular form: oligonucleotide

(xi) SEQ ID NO: SEQ ID NO: 18:

Claims (22)

An isolated polynucleotide comprising a component selected from the group consisting of (a) to (d): (a) a polynucleotide encoding a polypeptide comprising amino acids -21 to amino acid 99 of SEQ ID NO: 2; (b) a polynucleotide encoding a polypeptide comprising amino acids 1 to 99 of SEQ ID NO: 2; (c) a polynucleotide capable of hybridizing with the polynucleotide of (a) or (b) and at least 70% identical; (d) a polynucleotide fragment of the polynucleotide of (a) or (b). The polynucleotide of claim 1 wherein the polynucleotide is DNA. The polynucleotide of claim 1 wherein the polynucleotide is RNA. The polynucleotide of claim 1 wherein the polynucleotide is genomic DNA. 3. The polynucleotide of claim 2 encoding a polypeptide comprising amino acids -21-99 of SEQ ID NO: 2. 3. The polynucleotide of claim 2 encoding a polypeptide comprising amino acids 1 to 99 of SEQ ID NO: 2. An isolated polynucleotide comprising a component selected from the group consisting of (a) to (c): (a) a polynucleotide encoding a polypeptide having an amino acid expressed by a DNA contained in ATCC Accession No. 75676; (b) a polynucleotide capable of hybridizing with the polynucleotide of (a) and at least 70% identical; (c) a polynucleotide fragment of the polynucleotide of (a) or (b). The polynucleotide of claim 1 comprising the nucleotide 1 to nucleotide 363 sequences of SEQ ID NO: 1. A vector comprising a DNA as defined in claim 2. A host cell genetically engineered using the vector defined in claim 9. A method of producing a polypeptide comprising expressing a polypeptide encoded by said DNA from a host cell of claim 10. A method of producing a cell capable of expressing a polypeptide comprising a cell genetically engineered with the vector of claim 9. (i) a polypeptide having the amino acid sequence and fragments, analogs and derivatives thereof derived from SEQ ID NO: 2; And (ii) a polypeptide selected from the group consisting of a polypeptide and fragments, analogs and derivatives of said polypeptide encoded by the cDNA of ATCC accession No. 75676. The polypeptide of claim 13, wherein the polypeptide has an amino acid sequence derived from SEQ ID NO: 2. Agonists for the polypeptides as defined in claim 13. Antagonist to the polypeptide as defined in claim 13. A method of treating a patient in need of Ckβ-8, comprising administering to the patient a therapeutically effective amount of the polypeptide as defined in claim 13. The method of claim 17, wherein the therapeutically effective amount of the polypeptide is administered by providing a patient with DNA that encodes and expresses the polypeptide in vivo. A method of treating a patient in need of inhibition of Ckβ-8 polypeptide, comprising administering to the patient a therapeutically effective amount of the antagonist of claim 16. A method of diagnosing susceptibility to a disease or condition associated with expression of a polypeptide of claim 13 comprising determining a mutation in a nucleic acid sequence encoding the polypeptide. A diagnostic method comprising analyzing the presence of a polypeptide of claim 13 in a sample derived from a host cell. A cell separated from the polypeptide by a porous filter, a compound to be screened, and a combination of said polypeptides; And A method of identifying an antagonist and agonist for the polypeptide of claim 13, including determining the extent of movement of the cell to determine whether the compound is an effective antagonist or agonist.