JP2003093076A - HUMAN KEMOKINE beta-13 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a human kemokine polypeptide, and to provide a DNA (RNA) encoding the kemokine polypeptide. SOLUTION: A method for using the kemokine polypeptide isolated from recombination technology for the treatment of leukemia, tumor, chronic infectious diseases, autoimmune diseases, fibrotic diseases, wounds and psoriasis. An antagonist against the kemokine polypeptide. Their employment as therapeutic medicines for treating chronic rheumatoid arthritis, autoimmune diseases, allergic reactions, prostaglandin-independent fever, and bone marrow failure. A diagnostic assay for detecting the variation of a nucleic acid sequence and the change of a polypeptide concentration. The detection of a variation in a polynucleotide encoding the kemokine polypeptide. And a diagnostic assay for detecting the change of a polypeptide level in a host cell.

Description

Detailed Description of the Invention

The present invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, and the production of such polynucleotides and polypeptides. . More specifically, the polypeptides of the present invention have been putatively identified as human chemokine polypeptides, and are sometimes referred to hereinafter as human chemokine β-13 (Ckβ-13). The invention also relates to inhibiting the action of such polypeptides.

Chemokines, also called intercrine cytokines, are a subfamily of structurally and functionally related cytokines. These molecules are
The size is 8 to 10 kd. In general, chemokines show 20% -75% homology at the amino acid level and are characterized by four conserved cysteine residues that form two disulfide bonds. Based on the sequence of the first two cysteine residues, chemokines are classified into two subfamilies, α and β. In the α subfamily, the first two cysteines are separated by one amino acid and are therefore referred to as the “CXC” subfamily. In the β subfamily, the two cysteines are located in adjacent positions and are therefore referred to as the “CC” subfamily. Therefore, at least nine different members of this family have been identified in humans.

Intercrine cytokines exhibit a wide variety of functions. A prominent property is the ability to induce chemotactic migration of different cell types, including monocytes, neutrophils, T lymphocytes, basophils, and fibroblasts. Many chemokines have pro-inflammatory activity and are involved in many stages during the inflammatory response. These activities include stimulation of histamine release, release of lysosomal enzymes and leukotrienes, enhanced adhesion of target immune cells to endothelial cells, enhanced complement protein binding, induction of granulocyte adhesion molecule and complement receptor expression. , And respiratory burst. In addition to their situation in inflammation,
Certain chemokines have been shown to exhibit other activities. For example, macrophage inflammatory protein 1 (MIP-
1) can suppress the proliferation of hematopoietic stem cells, and induce platelet factor-4 (PF
-4) is a potent inhibitor of endothelial cell proliferation, interleukin-8 (IL-8) promotes keratinocyte proliferation, and GRO is an autocrine growth factor for melanoma cells.

In the light of their diverse biological activities, chemokines are found in many physiological and disease conditions (lymphocyte migration, wound healing, hematopoietic regulation, as well as allergies, asthma,
And their implications (including immunological injuries such as arthritis) are not surprising.

Members of the "CC" branch exert their effects on the following cells: eosinophils that destroy parasites, reduce parasitic infections, and cause chronic inflammation in the respiratory tract airways. Spheres; monocytes and macrophages that suppress tumor formation in vertebrates; T lymphocytes that attract T cells, and basophils that release histamine, which plays a role in allergic inflammation.

Members of the CC branch act predominantly in mononuclear cells and members of the CXC branch predominantly act in neutrophils, while different chemoattractant properties are assigned to chemokines based on this index. Cannot be done. Some chemokines from one family exhibit characteristics of the other.

The polypeptides of the present invention have a conserved cysteine "CC" region and have amino acid sequences homologous to known chemokines.

In accordance with one aspect of the present invention, there are provided novel polypeptides and biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.

According to another aspect of the present invention there is provided an isolated nucleic acid molecule encoding such a polypeptide, the nucleic acid molecule comprising mRNA, DNA, cDNA, genomic DNA, as well as biologically active And fragments thereof, analogs and derivatives thereof, which are useful for diagnosis or therapy.

According to another aspect of the invention, there is provided a nucleic acid probe comprising a nucleic acid molecule of sufficient length to specifically hybridize to a nucleic acid sequence encoding a polypeptide of the invention.

According to a still further aspect of the invention, a recombinant prokaryotic host cell comprising a nucleic acid sequence encoding a polypeptide of the invention under conditions which promotes expression of said protein and subsequent recovery of said protein, and Recombinant techniques that include culturing / or recombinant eukaryotic host cells provide the steps for producing such polypeptides.

According to yet a further aspect of the invention, for example, to treat solid tumors, chronic infections, leukemias, T cell mediated autoimmune diseases, parasitic infections, psoriasis, growth factors, growth factors. To utilize such a polypeptide or polynucleotide that encodes such a polypeptide for therapeutic purposes, for stimulating activity, for inhibiting angiogenesis, and for promoting wound healing. A process is provided.

According to yet a further aspect of the invention there is provided an antibody against such a polypeptide.

According to a still further aspect of the invention there is provided an antagonist to such a polypeptide, which is, for example, a particular autoimmune disease, atherosclerosis, chronic inflammatory and infectious disease, histamine. And IgE-mediated allergic reaction, prostaglandin-independent fever, bone marrow failure, silicosis, sarcoidosis,
It can be used to inhibit the action of such polypeptides in the treatment of rheumatoid arthritis and hypereosinophilic syndrome.

According to another aspect of the present invention, a method of diagnosing a disease or susceptibility to a disease associated with mutations in the nucleic acid sequences of the invention and altered levels of the protein encoded by such nucleic acid sequences. Methods are provided.

According to a still further aspect of the present invention, such a polypeptide, or a polynucleotide encoding such a polypeptide, is used for in vitro purposes related to scientific research, DNA synthesis and DNA vector production. A process is provided for utilization.

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

The drawings are illustrations of embodiments of the invention,
It is not meant to limit the scope of the invention, which is covered by the claims.

In accordance with one aspect of the present invention, the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2), or the clone deposited on April 28, 1995 as ATCC Deposit No. 97113. An isolated nucleic acid (polynucleotide) encoding a mature polypeptide encoded by the cDNA (s) is provided.

A polynucleotide encoding Ckβ-13 was isolated from an activated monocyte cDNA library. Ckβ
-13 is a member of the CC branch of chemokines. This is a putative leader sequence with approximately the first 28 amino acid residues, so that the mature protein contains 65 amino acids, 93
It contains an open reading frame encoding a protein of amino acid residues. The protein has structural homology to the chemokine polypeptide and 33% identity and 53% similarity to the human MIP-1α polypeptide, where the entire sequence is used alone as an example.

Four spatially conserved cysteine residues in chemokines are found in the polypeptides of the invention, as can be seen in FIG.

The polynucleotide of the present invention is in the form of RNA or DNA (this DNA can be cDNA, genomic DNA, and synthetic DN
Wrap A)). DNA can be double-stranded or single-stranded, and, if single-stranded, can be the coding strand or non-coding (antisense) strand. The coding sequence encoding the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID NO: 1) or the deposited clone (s), or the coding sequence is a duplication of the genetic code. Or as a result of degeneracy,
It may be a different coding sequence encoding the same mature polypeptide as the DNA of (SEQ ID NO: 1) or the deposited cDNA.

The polynucleotide encoding the mature polypeptide of Figure 1 (SEQ ID NO: 2) or the mature polypeptide encoded by the deposited cDNA may include: the coding sequence of the mature polypeptide only; of the mature polypeptide Coding sequences, and additional coding sequences such as leader or secretory sequences, or proprotein sequences; coding sequences for mature polypeptides (and optionally additional coding sequences) and non-coding sequences (eg, introns or mature polypeptides). 5'and / or 3'non-coding sequences of the coding sequence).

Accordingly, the term "polynucleotide encoding a polypeptide" refers to a polynucleotide containing only the coding sequence of the polypeptide, as well as additional coding sequences,
And / or a polynucleotide comprising non-coding sequences.

The present invention further encodes a polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2), or fragments, analogs and derivatives of the polypeptide encoded by the cDNA of the deposited clone (s). The present invention relates to variants of the polynucleotides described above. A variant of a polynucleotide can be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Accordingly, the invention provides a polynucleotide encoding the same mature polypeptide as shown in Figure 1 (SEQ ID NO: 2), or the same mature polypeptide encoded by the cDNA of the deposited clone (s), As well as variants of such polynucleotides. This variant encodes a fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO: 2) or the polypeptide encoded by the cDNA of the deposited clone (s). Such nucleotide variants include deletion variants, substitution variants, and addition or insertion variants.

As indicated herein above, the polynucleotide is the coding sequence shown in Figure 1 (SEQ ID NO: 1), or the coding sequence of the deposited clone (s).
It may have a coding sequence that is a naturally occurring allelic variant. As is known in the art, allelic variants are another form of polynucleotide sequence that may have one or more nucleotide substitutions, deletions or additions, which affect the function of the encoded polypeptide. Virtually unchanged.

The present invention also includes a polynucleotide,
Here, the coding sequence for the mature polypeptide is a polynucleotide sequence that assists in the expression and secretion of the polypeptide from the host cell in the same reading frame (e.g., to control the transport of the polypeptide from the cell, Leader sequence which functions as a secretory sequence). A polypeptide with a leader sequence is a preprotein and has a leader sequence that is cleaved by the host cell, and may form the mature form of the polypeptide. These polynucleotides also contain additional 5 '
It may encode a proprotein which is a mature protein plus amino acid residues. A mature protein with a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved, the active mature protein remains.

Therefore, for example, the polynucleotide of the present invention is a mature protein, or a protein having a pro sequence, or a pro sequence and a pre sequence (leader sequence).
A protein having both can be encoded.

The polynucleotide of the present invention may also have a coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexahistidine tag provided by the pQE-9 vector which, in the case of a bacterial host, provides for purification of the mature polypeptide fused to the marker. Alternatively, for example, the marker sequence is a mammalian host (e.g., COS
-7 cells) is used, it may be a hemagglutinin (HA) tag. The HA tag matches an epitope derived from the influenza hemagglutinin protein (Wilson, I. et al., Cel
l, 37: 767 (1984)).

The term "gene" means the segment of DNA involved in the production of polypeptide chains; that is, the regions preceding and following the coding region (leaders and trailers) as well as the individual coding segments (exons). Includes a sequence (intron) interposed between.

Fragments of the full-length Ckβ-13 gene are used to isolate the full-length gene and to isolate other genes with high sequence similarity to this gene or with similar biological activity. Can be used as a hybridization probe for a cDNA library. This type of probe preferably has at least 30 bases and may include, for example, 50 or more bases. This probe also corresponds to the full-length transcript cD
NA clones and complete Ckβ-13 containing regulatory and promoter regions, exons and introns
It can be used to identify genomic clone (s) containing the gene. As an example of screening, known DNA for synthesizing oligonucleotide probes
By using the sequence, it is possible to isolate the coding region of the Ckβ-13 gene. A labeled oligonucleotide having a sequence complementary to the sequence of the gene of the present invention is used to screen a human cDNA library, genomic DNA or mRNA to determine which member of the library this probe hybridizes to. used.

The invention further provides at least 70 between the sequences.
%, Preferably at least 90%, and more preferably at least 95% identity to polynucleotides that hybridize to the sequences herein above. The invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides herein above. The term "stringent conditions" as used herein means that hybridization will occur only if there is at least 95%, and preferably at least 97%, identity between the sequences. In a preferred embodiment, the polynucleotide that hybridizes to the polynucleotide herein above is the cDNA of Figure 1 (SEQ ID NO: 1) or the deposited cDNA.
Encodes a polypeptide that retains either substantially the same biological function or activity (ie, functions as a chemokine polypeptide) as the mature polypeptide encoded by (s).

Alternatively, the polynucleotide may be a polypeptide having at least 20 bases, preferably 30 bases, and more preferably at least 50 bases.
These hybridize to the polynucleotides of the invention and have identity thereto and may or may not retain activity, as described herein above. For example, such a polynucleotide may be used as a probe for the polynucleotide of SEQ ID NO: 1, or a variant thereof, for example, for recovery of the polynucleotide, or as a diagnostic probe, or as a PCR primer. .

Accordingly, the present invention provides at least 70% identity with a polynucleotide encoding the polypeptide of SEQ ID NO: 2 as well as fragments thereof, which fragments are at least 30 bases, and preferably 50 bases.
It preferably relates to polynucleotides having at least 90%, and more preferably at least 95% identity, and polynucleotides encoded by such polynucleotides.

Deposit (s) as used herein
Is maintained under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure. These deposits are
It is provided only as a convenience to those of ordinary skill in the art and is not an admission that a deposit is required under 35 USC 112. The sequences of the polynucleotides contained in the deposit, as well as the amino acid sequences of the polypeptides encoded thereby, are incorporated herein by reference,
It then controls the event of any conflicts with the description of sequences herein. A license may be required in order to manufacture, use, or sell the deposit, and such license is not hereby provided.

The present invention further includes a polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2) or having the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and fragments of such polypeptides. Regarding derivatives.

The terms "fragment,""derivative," and "analog" refer essentially to the polypeptide of Figure 1 (SEQ ID NO: 2) or the polypeptide encoded by the deposited cDNA. To a polypeptide possessing the same biological function or activity. Thus, analogs include proproteins that can be activated by cleavage of the proprotein portion to produce the active mature polypeptide. Derivatives or fragments can include splice variants that have, for example, fewer amino acid residues than the polypeptide of Figure 1, but still retain the biological activity characteristic of human chemokine polypeptides.

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

The fragment, derivative, or analog of the polypeptide of FIG. 1 (SEQ ID NO: 2) or the polypeptide encoded by the deposited cDNA has (i) one or more amino acid residues that are conservative amino acid residues or non- It is substituted with a conservative amino acid residue (preferably a conservative amino acid residue), and such substituted amino acid residue may or may not be an amino acid residue encoded by the genetic code. Good, or (ii) one or more amino acid residues containing substituents, or (iii) a compound in which the mature polypeptide increases the half-life of the polypeptide
Fused with another compound (e.g. polyethylene glycol), or (iv) additional amino acids
The (eg, leader sequence, or secretory sequence, or a sequence used to purify the mature polypeptide or proprotein sequence) may be fused to the mature polypeptide. Such fragments, derivatives and analogs are considered to be within the skill of those in the art given the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in isolated form and are preferably purified to homogeneity. The term "isolated"
Means that the material is removed from its original environment (eg, the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal has not been isolated, but is the same polynucleotide or polypeptide that is separated from some or all of the coexisting materials in the natural system. The polypeptide has been isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, and still such a vector or composition may be part of its native It is isolated in that it is not part of the environment.

Polypeptides of the invention include polypeptides of SEQ ID NO: 2 (particularly mature polypeptides), as well as polypeptides of SEQ ID NO: 2 of at least 70% similarity (preferably at least 70% identity), and more Preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO: 2, and even more preferably at least 95% similarity (even more preferably to the polypeptide of SEQ ID NO: 2). Is at least 95%
Identity) and also at least 30 amino acids, and more preferably at least
It includes a portion of such a polypeptide that typically contains 50 amino acids.

As is known in the art, "similarity" between two polypeptides means that the second
Of the polypeptide and its conservative amino acid substitutions.

Fragments or portions of the polypeptides of the present invention can be used to produce the corresponding full-length polypeptide by peptide synthesis. Therefore, this fragment can be used as an intermediate to produce a full-length polypeptide. Fragments or portions of the polynucleotides of the invention can be used to synthesize the full length polynucleotides of the invention.

The present invention also relates to vectors containing the polynucleotides of the invention, host cells genetically engineered with the vectors of the invention, and the production of polypeptides of the invention by recombinant techniques.

The host cell is the vector of the invention (which is
For example, genetically engineered (transduced, or can be a cloning vector or expression vector),
Or transformed or transfected). Vectors can be in the form of, for example, plasmids, virus particles, phages and the like. Engineered host cells can be cultured in conventional nutrient media appropriately modified to activate promoters, select for transformants, or amplify the Ckβ-13 gene. Culture conditions (eg, temperature, pH, etc.) are those previously used for the host cell selected for expression and will be apparent to those of skill in the art.

The polynucleotide of the present invention can be used to produce the polypeptide by recombinant techniques. Thus, for example, the polynucleotide can be included in any one of a variety of expression vectors for expressing the polypeptide. Such vectors include chromosomal DNA sequences, non-chromosomal DNA sequences, and synthetic DNA sequences. Such vectors include, for example, derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from a combination of plasmid and phage DNA, viral DNA (eg, vaccinia, adenovirus, fowlpox virus, And pseudorabies). However, any other vector may be used, so long as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence will be labeled with appropriate restriction endonuclease sites by procedures known in the art.
Inserted in (single or multiple). Such and other procedures are considered to be within the skill of those in the art.

The DNA sequence in the expression vector is operably linked to the appropriate expression control sequence (s) (promoter) to direct the synthesis of mRNA. Representative examples of such promoters may include: LTR
Or SV40 promoter, E. coli lac or trp, lambda phage P _L promoter, and prokaryotic or eukaryotic cells or other promoters known to control expression of genes in the virus. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

Furthermore, the expression vector preferably has phenotypic characteristics for the selection of transformed host cells (eg dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, or tetracycline in E. coli , for example). Resistance or ampicillin resistance).

Vectors containing a suitable DNA sequence as described herein above, as well as a suitable promoter or control sequence, can be used to transform a suitable host and express the protein in the host.

Representative examples of suitable hosts may include: bacterial cells (eg E. coli , Streptomyce).
s , Salmonella typhimurium ); fungal cells (eg yeast);
Insect cells (e.g. Drosophila S2 and Spodoptera Sf
9 ); animal cells (eg, CHO, COS or Bowes melanoma);
Adenovirus; plant cells, etc. Selection of a suitable host is
It is believed to be within the skill of those in the art given the teachings herein.

More particularly, the present invention also encompasses recombinant constructs containing one or more sequences as broadly described above.
The construct includes a vector (e.g., a plasmid vector or a viral vector), in which
The sequences of the invention are inserted in either the forward or reverse orientation.
In a preferred aspect of this embodiment, the construct further comprises a regulatory sequence operably linked to the sequence (eg, encasing a promoter). Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided as examples. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD1
0, phagescript, psiX174, pBluescript SK, pBSKS, pN
H8A, pNH16a, pNH18A, pNH46A (Stratagene); pTRC99a,
pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, p0G44, pXT1, pSG (Stratagen
e), pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

The promoter region can be selected from any desired gene using the CAT (chloramphenicol transferase) vector or other vector carrying a selectable marker. Two suitable vectors are pKK232
-8 and pCM7. Particularly well known bacterial promoters are lacI, lacZ, T3, T7, gpt, λP _R , P _L and t
rp is an example. Eukaryotic promoters are CMV immediate type, HSV thymidine kinase, early SV40 and late SV40,
Includes retrovirus-derived LTRs, and mouse metallothionein I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described construct. The host cell can be a higher eukaryotic cell (eg, a mammalian cell) or a lower eukaryotic cell (eg, a yeast cell), or the host cell can be a prokaryotic cell (eg, a bacterial cell). Introduction of the construct into the host cell can be achieved by calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation (Davi
s, L., Dibner, M., Battey, I., Basic Methods in Molecu
lar Biology, 1986).

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention may be synthetically produced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast,
It can be expressed in bacteria, or other cells under the control of a suitable promoter. Cell-free translation systems can also be used to produce such proteins using RNA derived from the DNA constructs of the invention. Suitable cloning and expression vectors used in prokaryotic and eukaryotic hosts are found in Sambrook et al., Molecular Cloning:
A Laboratory Manual, 2nd Edition, Cold Spring Harbor,
NY, (1989), the disclosure of which is incorporated herein by reference.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancer
A cis-acting element of DNA, usually about 10 to about 300 bp
Which acts on the promoter to increase its transcription. As an example, SV on the late side of the replication origin bp 100-270
40 enhancers, early promoter enhancers of cytomegalovirus, polyoma enhancers on the late side of the replication origin, and adenovirus enhancers.

In general, recombinant expression vectors contain origins of replication and selectable markers that enable the transformation of host cells.
(For example, the E. coli ampicillin resistance gene, and Sc
erevisiae TRP1 gene), as well as a promoter from a highly expressed gene that directs transcription of downstream structural sequences.
Such a promoter may be derived from an operon encoding a glycolytic enzyme (eg, 3-phosphoglycerate kinase (PGK)), α factor, acid phosphatase, heat shock protein, or the like. Heterologous structural sequences are assembled in proper phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence has the desired characteristics (e.g.,
A fusion protein can be encoded that includes an N-terminal identification peptide that provides for stabilization or simplified purification of the expressed recombinant product.

Expression vectors useful for bacterial use are constructed by inserting a structural DNA sequence encoding the desired protein, with appropriate translation initiation and termination signals, in operable reading phase with a functional promoter. To be done. The vector contains one or more phenotypic selectable markers, and an origin of replication to ensure maintenance of the vector and optionally provide amplification in the host. Suitable prokaryotic hosts for transformation are E. coli ,
Bacillus subtilis , Salmonella typhimurium , and Pseudomonas, Streptomyces, and Staphylococ
Includes various species within the cus genus, but other species may also be used for selection.

As a representative, but non-limiting example, expression vectors useful for bacterial use include selectable markers and bacterial replication derived from commercially available plasmids containing the genetic elements of the well known cloning vector pBR322 (ATCC 37017). The origin may be included. Such commercially available vectors are described, for example, in pKK223-3 (Pharmacia Fine Chemicals, Upp.
sala, Sweden) and pGEM1 (Promega Biotec, Madison,
WI, USA). These pBR322 "skeletons" are
Combined with a suitable promoter and the structural sequence to be expressed.

Following transformation of the appropriate host strain and growth of the host strain to the appropriate cell density, the selected promoter is induced by appropriate means (eg temperature shift or chemical induction) and the cells Incubate for a further period.

The cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells used in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those of skill in the art.

A variety of mammalian cell culture systems can also be used to express recombinant protein. Examples of mammalian expression systems include the COS-7 strain of monkey kidney fibroblasts described in Gluzman, Cell, 23: 175 (1981), and other cell lines capable of expressing compatible vectors (eg, C127, 3T3,
CHO, HeLa, and BHK cell lines). Mammalian expression vectors include origins of replication, suitable promoters and enhancers, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and splice acceptor sites, transcription termination sequences, and 5'flanking nontranscribed sequences. including. DNA sequences derived from the SV40 splice site and polyadenylation site are
It can be used to provide the required nontranscribed genetic elements.

Polypeptides may be ammonium sulfate precipitated or ethanol precipitated, acid extracted, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Can be recovered from recombinant cell culture and purified by methods including. If desired, protein refolding steps can be used to complete the alignment of the mature protein. Finally, high performance liquid chromatography (HPLC)
Can be used in the final purification step.

The polypeptide of the present invention may be a naturally purified product, or the product of a chemical synthetic procedure, or may be a prokaryotic or eukaryotic host (eg, bacteria, yeast, higher in culture, etc.). (By plant, insect, and mammalian cells) by recombinant techniques. Depending on the host used in a recombinant production procedure, the polypeptides of the present invention may or may not be glycosylated. The polypeptides of the present invention may also include an initial methionine amino acid residue.

The polynucleotides and polypeptides of the present invention can be used as research reagents and materials for discovery of treatments and diagnostics to human disease.

The polypeptides of the present invention may be used to inhibit bone marrow stem cell colony formation as an adjunctive protective treatment during cancer chemotherapy. Ckβ-13 polypeptides can inhibit the proliferation and differentiation of hematopoietic cells such as bone marrow stem cells.
Progenitor cells that are committed to differentiation (e.g., granulocytes,
And the inhibitory effect on the population of macrophages / monocytes) could be used therapeutically to inhibit the growth of leukemic cells.

The polypeptides of the present invention inhibit the proliferation of epidermal keratinocytes for the treatment of psoriasis, which is characterized by keratinocyte hyperproliferation, because Langerhans cells in the skin have been found to produce chemokines. Can be used to use.

The polypeptides of the present invention may also be used in host defense cells such as cytotoxic T cells and macrophages.
Can be used to treat solid tumors, such as Kaposi's sarcoma, by stimulating the invasion and activation of B. elegans via chemotaxis and by inhibiting tumor angiogenesis.

They may also be used to enhance the host's defense against resistant chronic and acute infections (eg Mycobacterium infection) through the attraction and activation of bactericidal leukocytes.

The polypeptides of the present invention may also be used to inhibit T cell proliferation by inhibiting IL-2 biosynthesis for the treatment of T cell mediated autoimmune diseases and lymphocytic leukemia. Can be done.

Ckβ-13 also stimulates wound healing, both through clearing debris and recruiting connective tissue-promoting inflammatory cells, and also controlling excessive TGF-β-mediated fibrosis. And can be used to prevent scarring during wound healing. In this same manner, Ckβ-13 can also be used to treat cirrhosis, osteoarthritis, and other fibrotic disorders including pulmonary fibrosis.

The polypeptides of the present invention also increase the presence of eosinophils, which have the unique function of killing parasite larvae that invade tissues, such as in schistosomiasis, trichinellosis, and ascariasis. .

They can also be used to regulate hematopoiesis by regulating the activation and differentiation of various hematopoietic progenitor cells, for example to release mature leukocytes from the bone marrow after chemotherapy.

Using the polypeptides of the invention,
Unwanted cells may be targeted for apoptosis, for example in the treatment of cancer.

Polynucleotides and polypeptides encoded by such polynucleotides may also be used for in vitro purposes related to scientific research, DNA synthesis, and DNA vector production, as well as for the treatment of human disease. Available for the design of treatments and diagnostics for.

The polypeptide may also be used to recruit bone marrow stem cells into peripheral blood, which facilitates isolation of stem cells. The isolated stem cells can be used to colonize bone marrow after high dose chemotherapy.

The present invention also relates to the Ckβ-13 gene as part of a diagnostic assay for detecting a disease associated with the presence of a mutation in a nucleic acid sequence encoding a polypeptide of the invention or susceptibility to such a disease. Regarding use. Such diseases (eg, tumors and cancers) are associated with underexpression of human chemokine polypeptides.

Individuals carrying mutations in the gene of the present invention can be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis can be obtained from cells of patients such as blood, urine, saliva, tissue biopsies and autopsy materials.
Genomic DNA can be used directly for detection or PCR (Saiki et al., Nature, 324: 163-166 (198) prior to analysis.
6)) can be used to enzymatically amplify. RNA or cDNA may also be used for the same purpose. As an example, Ckβ
PCR primer complementary to the nucleic acid encoding -13 is Ckβ
-13 can be used to identify and analyze mutations. For example, deletions and insertions can be detected by a change in the size of the amplification product in comparison to the normal genotype. Point mutation causes Ck to radiolabel the amplified DNA
It can be identified by hybridizing to β-13 RNA, or radiolabeled Ckβ-13 antisense DNA sequence. A perfectly matched sequence can be distinguished from an unmatched duplex by RNase A digestion or by a different melting point.

Genetic testing based on DNA sequence differences can be accomplished by detecting alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing reagents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DN with different sequence
A fragments can be distinguished on denaturing formamide gradient gels in which the mobility of different DNA fragments is delayed to different positions in the gel according to their specific or partial melting points (eg Myers et al., Science, 23.
0: 1242 (1985)).

Sequence changes at specific positions also resulted in RNas
e, and S1 protection, or nuclease protection assays such as chemical cleavage methods (eg, Cotton et al., PNAS, USA, 8
5: 4397-4401 (1985)).

Therefore, detection of specific DNA sequences can be performed by hybridization, RNase protection, chemical cleavage, direct
It may be accomplished by methods such as DNA sequencing, or the use of restriction enzymes (eg, restriction fragment length polymorphism analysis (RFLP)), and Southern blots of genomic DNA.

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

The present invention also provides the polypeptide of the present invention in a variety of tissues, as overexpression of the polypeptide as compared to normal control tissue samples can detect the presence or susceptibility to a disease such as a tumor. To a diagnostic assay for detecting changes in the levels of
Assays used to detect levels of a polypeptide of the invention in a sample from a host are well known to those of skill in the art and include such assays as radioimmunoassays, competitive binding assays, Western blot analysis,
Included are ELISA assays and "sandwich" assays. ELISA assay (Coligan et al., Current Protocols
in Immunology, 1 (2), Chapter 6, (1991)), first, C
It includes the step of preparing an antibody (preferably a monoclonal antibody) specific for the kβ-13 antigen. In addition, a reporter antibody is prepared against the monoclonal antibody. The reporter antibody is conjugated with a radioactive, fluorescent, or detectable reagent, such as horseradish peroxidase enzyme in this example. The sample is removed from the host and incubated on a solid support (eg polystyrene dish) that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as BSA. The monoclonal antibody is then incubated in the dish during which time the monoclonal antibody binds to any Ckβ-13 protein bound to the polystyrene dish. All unbound monoclonal antibody is removed by washing with buffer. The horseradish peroxidase-conjugated reporter antibody, in turn, is placed in the dish so that the reporter antibody binds to any monoclonal antibody bound to the Ckβ-13 polypeptide. The unbound reporter antibody is then washed away. The peroxidase substrate is then added to the dish, and the amount of color development within a given time period is a measure of the amount of Ckβ-13 protein present within a given dose of the patient sample when compared to the standard curve. .

A competition assay may be used, where Ckβ
-13 polypeptide-specific antibody is bound to a solid support, and labeled Ckβ-13 polypeptide and a sample from the host pass through the solid support and, for example,
The amount of label detected by liquid scintillation chromatography can be correlated to the amount of Ckβ-13 polypeptide in the sample.

The "sandwich" assay is similar to the ELISA assay. In the "sandwich" assay, the Ckβ-13 polypeptide passes through the solid support and binds to the antibody bound to the solid support. Then 2
The secondary antibody binds to the Ckβ-13 polypeptide. A tertiary antibody, which is labeled and specific for the secondary antibody, can then be passed through the solid support and bound to the secondary antibody, and the amount quantitated.

The present invention provides methods for identifying receptors for the polypeptides of the present invention. Genes encoding receptors are described, for example, in ligand panning and FACS sorting (Coligan et al., Current Protocol.
s in Immun., 1 (2), Chapter 5, (1991)) can be identified by a number of methods known to those skilled in the art. Preferably, expression cloning is used where polyadenylated RNA is prepared from cells responsive to the polypeptide,
The cDNA library made from this RNA is then divided into pools and used to transfect COS cells or other cells that do not respond to this polypeptide. The transfected cells grown on glass slides are exposed to the labeled polypeptide. The polypeptide can be labeled by various means, including iodination or encapsulation of recognition sites for site-specific protein kinases. Following fixation and incubation,
The slides are submitted for autoradiographic analysis. Positive pools were identified, and the subpools were prepared and retransfected using the enterative subpooling and rescreening steps, finally resulting in a single clone (single or single) encoding the putative receptor. Multiple).

As an alternative approach for receptor identification, labeled polypeptides can be photoaffinity-linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material was resolved by PAGE analysis and X
Expose to line film. The labeled complex containing the receptor for the polypeptide can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing is used to design a set of degenerate oligonucleotide probes for screening a cDNA library to identify the gene encoding the putative receptor.

The present invention provides methods of screening compounds to identify agonists and antagonists to the polypeptides of the present invention. An agonist is a compound that binds to and activates a receptor for the polypeptide of the invention, while an antagonist is
Ckβ-13 binds to and and inhibits such receptors or simply for such receptors
Compete with. Chemotaxis can be assayed by placing cells chemoattracted by a polypeptide of the invention on a filter with pores of sufficient diameter (about 5 μm) to contain the cells. A solution of the potential agonist is placed in the lower part of the chamber with the appropriate control medium in the upper compartment, thus providing a gradient of agonist concentration over time in or through the porous membrane. It is measured by counting migrating cells.

When assaying for antagonists, the polypeptides of the invention are placed in the lower chamber and potential antagonists are added to determine if cell chemotaxis is inhibited.

Alternatively, a mammalian cell or membrane preparation expressing the receptor for this polypeptide is incubated with a labeled (eg radioactive) polypeptide of the invention in the presence of this compound. The ability of this compound to block this interaction is then measured.

Examples of potential antagonists to a polypeptide of the invention include antibodies, or in some cases oligonucleotides, which bind to this polypeptide. Another example of a potential antagonist is a negative dominant variant of this polypeptide. A negative dominant variant is a polypeptide that binds to the receptor for the wild-type polypeptide but retains no biological activity.

Antisense constructs prepared using antisense technology are also potential antagonists. Antisense technology can be used to control triplex helix formation, or expression of genes mediated by antisense DNA or RNA, both methods based on the binding of polynucleotides to DNA or RNA. For example, the 5'coding portion of the polynucleotide sequence encodes the mature polypeptide of the invention and is used to design an antisense RNA oligonucleotide of about 10-40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of a gene involved in transcription (triple chain helix, Lee et al., Nucl. Acids Res., 6: 3073 (1979); Coo
ney et al., Science, 241: 456 (1988); and Dervan et al., Sci.
ence, 251: 1360 (1991)). Thereby preventing production and production of the polypeptides of the invention. Antisense RNA oligonucleotides are
And block the translation of the mRNA molecule into a polypeptide (antisense-Okano, J. Neuro.
chem., 56: 560 (1991); oligodeoxynucleotides as antisense inhibitors of gene expression, CRC Pr
ess, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells such that antisense RNA or DNA can be expressed in vivo to inhibit production of the polypeptides of the invention.

Another potential antagonist is a peptide derivative of this polypeptide, which is a naturally or synthetically modified analog of this polypeptide that has lost biological function but is , Recognizes and binds to the receptor for this polypeptide and therefore effectively blocks this receptor. Examples of peptide derivatives include, but are not limited to, small peptides or peptide-like molecules.

Antagonists have been used to in certain autoimmune and chronic inflammatory and infectious diseases, macrophages and their precursors, as well as neutrophils, basophils, B lymphocytes and some T's. Cell subsets (eg activated T cells and CD8 cytotoxic T cells
Cells, as well as the chemotaxis and activation of natural killer cells). Examples of autoimmune diseases include multiple sclerosis, and insulin-dependent diabetes mellitus.

The antagonists may also be used to treat infectious diseases, including chylous embryosis, sarcoidosis, idiopathic pulmonary fibrosis, by interfering with mononuclear phagocyte recruitment and activation. They can also be used to treat idiopathic eosinophilia syndrome by inhibiting eosinophil production and migration. Endotoxic shock also
It can be treated with antagonists by inhibiting the migration of macrophages and their production of the polypeptides of the invention.

The antagonist may also be used to treat atherosclerosis by preventing the infiltration of monocytes into the arterial wall.

This antagonist also inhibits chemokine-induced mast cell and basophil degranulation and histamine release, thereby causing histamine-mediated allergic reactions and immunological disorders (late allergic reactions, chronic urticaria, and atopic reactions). (Including dermatitis). IgE-mediated allergic reactions such as allergic asthma, rhinitis, and eczema can also be treated.

The antagonist may also be used to treat chronic and acute inflammation by preventing the attraction of monocytes to the wound area. They also show that chronic and acute inflammatory lung disease causes retention of mononuclear phagocytes in the lung.
Being associated with sequestration, it can be used to regulate the macrophage population in normal lung.

Antagonists can also be used to treat rheumatoid arthritis by preventing the attraction of monocytes to the synovial fluid in the joints of patients. Monocyte influx and activation play important roles in the pathogenesis of both degenerative and inflammatory arthritis.

This antagonist can be used to block the deleterious cascade primarily attributable to IL-1 and TNF, which blocks the biosynthesis of other inflammatory cytokines. In this way, the antagonist can be used to prevent inflammation. This antagonist may also be used to inhibit chemokine-induced prostaglandin-independent fever.

The antagonist may also be used to treat cases of bone marrow failure, such as aplastic anemia and myelodysplastic syndrome.

The antagonist may also be used to treat asthma and allergies by preventing the accumulation of eosinophils in the lung. The antagonist may also be used to treat subepithelial basement membrane fibrosis, a hallmark of asthmatic lungs.

The antagonist can be used, for example, in a composition with a pharmaceutically acceptable carrier as described herein below.

The polypeptides of the present invention, as well as agonists and antagonists, can be used in combination with a suitable pharmaceutical carrier. Such compositions contain a therapeutically effective amount of the polypeptide and a pharmaceutically acceptable carrier or excipient. Such carriers include saline, buffered saline, dextrose,
Examples include, but are not limited to, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The present invention also provides one of the pharmaceutical compositions of the present invention.
Provided is a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients. Attached to such container (s) may be a form of notice set by a government agency that regulates the manufacture, use, or sale of pharmaceutical or biological products. The document reflects approval by the agency of manufacture, use, or sale for human administration. Moreover, the polypeptides, as well as agonists and antagonists, can be used in connection with other therapeutic compounds.

The pharmaceutical composition may be topical, intravenous, intraperitoneal,
It may be administered in a convenient manner such as by intramuscular, intratumoral, subcutaneous, intranasal, or intradermal routes. The pharmaceutical composition is administered in an effective amount for the treatment and / or prevention of the particular condition. Generally, the polypeptide is administered in an amount of at least about 10 μg / kg body weight, and most often in an amount not exceeding about 8 mg / kg body weight / day. In most cases, the dose is about 10 μg / kg to about 1 mg / kg body weight daily, taking into consideration the route of administration, symptoms and the like.

The polypeptides of the present invention, and agonists or antagonists that are polypeptides, can be used in accordance with the present invention by in vivo expression of such polypeptides, and this is often referred to as "gene therapy."

Thus, for example, a patient-derived cell may be prepared ex vivo using a polynucleotide (D
NA or RNA) and then provide the engineered cells to the patient to be treated with this polypeptide. Such methods are well known in the art. For example, cells can be engineered by procedures known in the art by use of retroviral particles containing RNA encoding the polypeptides of the present invention.

Similarly, cells may be engineered in vivo for expression of the polypeptide in vivo, eg, by procedures known in the art. As is known in the art,
Producer cells that produce retroviral particles that include RNA encoding a polypeptide of the invention can be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering the polypeptides of the present invention by such methods should be apparent to those of skill in the art from the teachings of the present invention. For example, the expression vehicle for manipulating cells can be other than a retrovirus, eg, an adenovirus that can be used to manipulate cells in vivo after combination with a suitable delivery vehicle.

As retroviruses from which the retroviral plasmid vector described above can be derived, Moloney murine leukemia virus, spleen necrosis virus, retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, gibbon leukemia virus,
Human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, breast cancer virus, and the like,
It is not limited to these. In one embodiment, the retroviral plasmid vector is from Moloney murine leukemia virus.

The vector contains one or more promoters. Suitable promoters that can be used include retroviral LTR; SV40 promoter, and human cytomegalovirus (CMV) promoter (Miller et al., Biotechnique
s , Vol. 7, No. 9, 980-990 (1989)), or any other promoter (eg, cellular promoters such as eukaryotic promoters (histone, pol III, and β-actin promoters. Including, but not limited to))). Other viral promoters that may be used include, but are not limited to, the adenovirus promoter, the thymidine kinase (TK) promoter, and the B19 parvovirus promoter. Selection of the appropriate promoter will be apparent to those of skill in the art from the teachings contained herein.

The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters that can be used include adenovirus promoters such as the adenovirus major late promoter; or heterologous promoters such as the cytomegalovirus (CMV) promoter; respiratory syncytial virus (RSV).
Promoter; inducible promoter such as MMT promoter, metallothionein promoter; heat shock promoter; albumin promoter; ApoAI promoter; human globin promoter; viral thymidine kinase promoter such as herpes simplex thymidine kinase promoter; retrovirus LTR (the above modified retrovirus (Including LTR); β-actin promoter; and human growth hormone promoter. The promoter can also be a naturally occurring promoter that controls the gene encoding the polypeptide.

Retroviral plasmid vectors can be used to transduce packaging cell lines to form producer cell lines. As an example of a packaging cell line that can be transfected, PE50
1, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H
2, ψCRE, ψCRIP, GP + E-86, GP + envAm12, and DAN cell line (Miller, Humman Gene Therapy , Volume 1, pages 5-14 (1
990)) and are not limited thereto, and these are incorporated herein by reference in their entirety. The vector may transduce the packaging cells by any means known in the art. Such means, electroporation, the use of liposomes, and CaPO ₄ precipitation include, but are not limited to. In another means, the retroviral plasmid vector can be encapsulated in liposomes or combined with lipids and then administered to the host.

The producer cell line produces infectious retroviral vector particles which contain the nucleic acid sequence (s) encoding this polypeptide. Such retroviral vector particles can then be used to transduce eukaryotic cells, either in vitro or in vivo. Transduced eukaryotic cells express the nucleic acid sequence (s) encoding this polypeptide. As eukaryotic cells that can be transduced, embryonic stem cells,
Embryonic cancer cells, as well as, but not limited to, hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells and bronchial epithelial cells.

The sequences of the present invention are also useful for chromosome identification. This sequence can be specifically targeted to and hybridized with a particular location on an individual human chromosome. Furthermore, there is currently a need to identify specific sites on the chromosome. Chromosomal marking reagents based on actual sequence data (repeated polymorphisms) are currently rarely used to mark chromosomal locations. The mapping of DNA to the chromosomes of the present invention is an important first step in relating these sequences to genes associated with disease.
It is a stage.

Briefly, the sequences can be mapped to the chromosome by the preparation of PCR primers (preferably 15-25 base pairs) from the cDNA. Computer analysis of the 3'untranslated region rapidly selects primers that span one or more exons in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids
It is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primer, sublocalization is
In a similar fashion, it can be achieved with a panel of fragments from a particular chromosome or pool of large genomic clone (s). Other mapping strategies that may also be used to map to that chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization with a constructed chromosome-specific cDNA library. To be

Fluorescence in situ hybridization (FISH) to the spread of metaphase nucleic acid (s) of cDNA clone (s) can be used to provide precise chromosomal localization in one step. This technology is 500
Alternatively, it can be used with a short cDNA of 600 bases. For a review of this technology, see Verma et al., Human Chromosomes: a.
Manual of Basic Techniques, Pergamon Press, New Y
See ork (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated to genetic map data. Such data can be found, for example, in V. McKusick, Mendelian Inheritance in Man (Johns
(Available online at Hopkins University Welch Medical Library). Then, the relationship between the gene mapped to the same chromosomal region and the disease is
Identified by linkage analysis (co-inheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is found in some or all of the affected individuals but not in normal individuals, then the mutation appears to cause the disease.

Using the resolution of current physical and genetic mapping techniques, the cDNA precisely located in the chromosomal region associated with disease is one of the potentially causative genes between 50 and 500. We obtain (this assumes 1 megabase mapping resolution and 1 gene per 20 kb).

The polypeptides, their fragments or other derivatives, or their analogs, or cells expressing them can be used as immunogens to produce antibodies to them. These antibodies can be, for example, polyclonal antibodies or polyclonal antibodies. The invention also provides chimeric, single-stranded,
And humanized antibodies, as well as Fab fragments,
Alternatively, it contains the products of the Fab expression library. Various procedures known in the art can be used for the production of such antibodies and fragments.

Antibodies raised against a polypeptide corresponding to a sequence of the invention may be obtained by direct injection of the polypeptide into an animal or by administration of the polypeptide to an animal, preferably a nonhuman animal. Can be done. The antibody so obtained is then itself bound to the polypeptide. In this manner, even sequences encoding only fragments of this polypeptide can be used to produce antibodies that bind whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from tissues that express it.

For the preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. As an example, hybridoma technology
(Kohler and Milstein, 1975, Nature, 256: 495-49.
7), trioma technology, human B cell hybridoma technology (Kozbor et al., 1983, Immunology Today 4:72), and EBV hybridoma technology for making human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibod).
ies and Cancer Therapy, Alan R. Liss, Inc., 77-96
Page).

The techniques described for the production of single chain antibodies (US Pat. No. 4,946,778) can be adapted to produce single chain antibodies to the immunogenic polypeptide products of this invention. Also, transgenic mice can be used to express humanized antibodies to immunogenic polypeptide products of this invention.

The present invention is further described with reference to the following examples; however, it should be understood that the invention is not limited to such examples. All parts or amounts are by weight unless otherwise noted.

In order to facilitate understanding of the following examples,
Describe certain frequently occurring methods and / or terms.

"Plasmid" is designated by a lowercase letter p and / or followed by uppercase letters and / or numbers. All of the starting plasmids herein are commercially available, publicly available on a non-limiting basis, or can be constructed from available plasmids by published procedures. Furthermore, equivalent plasmids to the described plasmids are known in the art,
And will be apparent to those skilled in the art.

"Digestion" of DNA refers to enzymatic cleavage of DNA with a restriction enzyme that acts only on specific sequences in the DNA.
Various restriction enzymes used herein are commercially available, and their reaction conditions, cofactors, and other requirements were used as known to those of skill in the art. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer. For the purpose of isolating DNA fragments for plasmid construction, typically 5-50 μg of DNA is digested with 20-250 units of enzyme in a larger dose. Appropriate buffer and substrate amounts for a particular restriction enzyme are specified by the manufacturer. About 1 hour, 37 ℃
Incubation times are normally used, but can be varied according to the supplier's instructions. After digestion, the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments gives
Performed using an 8% polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8: 4057 (1980).

An "oligonucleotide" is a single-stranded polydeoxynucleotide that can be chemically synthesized, or 2
Refers to any of the two complementary polydeoxynucleotide chains. Such synthetic oligonucleotides do not have a 5'phosphate and therefore will not bind to other oligonucleotides without the addition of phosphate with ATP in the presence of the kinase. Synthetic oligonucleotides bind to fragments that have not been dephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds between double-stranded nucleic acid fragments (Maniati
s, T. et al., Id., p. 146). Unless otherwise provided, the binding is 10 units of T4 DNA ligase ("ligase") per approximately equimolar amount of 0.5 μg of the DNA fragment to be ligated.
Can be achieved using known buffers and conditions.

Unless otherwise stated, transformation was by Graha
m, F. and Van der Eb., A., Virology, 52: 456-457 (1
973).

Example 1 Bacterial Expression and Purification of Ckβ-13 DNA Sequence Encoding Ckβ-13 (ATCC Deposit No. 97113)
Is the P'corresponding to the 5'and 3'terminal sequences of the processed Ckβ-13 nucleic acid sequence (excluding the putative signal peptide sequence).
Amplify first with CR oligonucleotide primers. Additional nucleotides corresponding to the Ckβ-13 gene were added at the 5'and 3'ends, respectively. The 5'oligonucleotide primer has the sequence 5'CCCGCATGCCCAACATG
16-nucleotide Ckβ-13 having GAAGACAG3 ′ (SEQ ID NO: 3), starting from the second nucleotide of the sequence encoding SphI restriction enzyme (bold) and the mature protein
Contains coding sequences. The ATG codon is contained at the SphI site. In the next codon following ATG, the first base is Sph
It is from the I site, and the remaining two bases correspond to the second and third bases of the first codon (residue 29) of the putative mature protein. 3'sequence, Contains a sequence complementary to the BamH1 site (bold) and is followed by 18 nucleotides of gene-specific sequence preceding the stop codon. The restriction enzyme site is the bacterial expression vector pQE-9
(Qiagen, Inc. Chatsworth, CA) corresponds to the restriction enzyme sites. pQE-9 is an antibiotic resistance (Amp ^r ), bacterial origin of replication (ori), IPTG regulatable promoter operator (P /
O), ribosome binding site (RBS), 6-His tag, and restriction enzyme site. Then pQE-9 was added to SphI and Bam
Digest with H1. The amplified sequence is ligated into pQE-9 and inserted in frame with the sequence encoding the histidine tag and RBS. The ligation mixture is then used to transform E. coli strain M
15 / rep4 (Qiagen, Inc.) to Sambrook, J. et al., Molecular Cl.
oning: ALaboratory Manual, Cold Spring LaboratoryP
Transform by the procedure described in ress, (1989). M15 / re
p4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kan ^r ). Transformants are identified by their ability to grow on LB plates and ampicillin / kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction enzyme analysis. The clone (s) containing the desired construct were designated as Amp (100 μg / ml).
Grow overnight (O / N) in liquid culture in LB medium supplemented with both Kan (25 μg / ml). 1: 100 with O / N culture
Inoculate large cultures at a ratio of ~ 1: 250. Cells are grown to an optical density of ⁶⁰⁰ (OD ⁶⁰⁰ ) of between 0.4 and 0.6. IPTG ("Isopropyl-BD-thiogalactopyranoside") is then added to a final concentration of 1 mM. IPTG
Activates the P / O by inactivating the lacI repressor.
To induce an increase in gene expression. Allow cells to grow for an additional 3-4 hours. The cells are then harvested by centrifugation. Cell pellets for chaotropic drug 6
Solubilize at pH 5.0 in M guanidine HCl. After clarification, the solubilized Ckβ-13 is purified from this solution by chromatography on a nickel-chelate column under conditions that allow for tight binding by proteins containing the 6-his tag (Hochuli, E et al., J. ．Chromatography 411: 177-184
(1984)). Ckβ-13 (greater than 98% pure) is eluted from the column with 6M guanidine HCl. Refolding of proteins except GnHCl can be accomplished by several protocols (J
aenicke, R. and Rudolph, R., Protein Structure-A
Practical Approach, IRL Press, New York (1990)). First, a dialysis step is utilized to remove GnHCl. Alternatively, the purified protein isolated from the Ni chelate column is
It can be bound to a second column that goes through a decreasing linear GnHCl gradient. Regenerates the protein while bound to the column,
Then 250 mM imidazole, 150 mM NaCl, 25 mM pH
Elute with a buffer containing 7.5 Tris HCl and 10% glycerol. Finally, the soluble protein is dialyzed against storage buffer containing 5 mM ammonium bicarbonate.

Example 2 Expression of Recombinant Ckβ-13 in COS Cells Expression of the plasmid, Ckβ-13 HA, was performed using the vector pcDNAI / Amp.
(Invitrogen). The vector pcDNAI / Amp contains: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E. origin of replication, 4) polylinker region, SV40
CMV promoter followed by introns and polyadenylation sites. The complete Ckβ-13 precursor, and the DNA fragment encoding the HA tag fused in frame to its 3'end, is cloned into the polylinker region of the vector. Therefore, recombinant protein expression is directed under the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H. Niman, R. Heighten, A Che.
renson, M.A. Connolly, and R. Lerner, 1984, Cell 3
7,767). The fusion of HA tag to the target protein results in HA
It allows easy detection of recombinant proteins using antibodies that recognize the epitope. The plasmid construction strategy is described below: Ckβ-1
The DNA sequence encoding 3 (ATCC Deposit No. 97113)
Construct by PCR with one primer: Contains a HindIII site followed by 18 nucleotides of the Ckβ-13 coding sequence starting at position 3 minus the start codon; (SEQ ID NO: 6) is an XbaI site, translation stop codon (underlined), HA
It contains a tag and a sequence complementary to the last 21 nucleotides (not including the stop codon) of the Ckβ-13 coding sequence. Therefore, the PCR product was fused in-frame with the HindIII site.
It contains a Ckβ-13 coding sequence followed by an HA tag, a translation stop stop codon adjacent to the HA tag, and an XbaI site. PCR amplified DNA
Fragment and vector (pcDNA3 / Amp) with HindIII
And digested with XbaI restriction enzyme and ligated. The ligation mixture was transformed into E. coli SURE strain (Stratagene Cloning Syst
ems, La Jolla, CA), inoculate the transformed culture on ampicillin medium plates, and select resistant colonies. Isolation of the plasmid DNA from the transformant,
The presence of the correct fragment is then tested by restriction analysis. For expression of recombinant Ckβ-13 polypeptide, COS cells are transfected with the expression vector by the DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Mol.
ecular Cloning: A Laboratory Manual, Cold Spring L
aboratory Press, (1989)). Expression of Ckβ-13 HA protein is detected by radiolabeling and immunoprecipitation.
(E. Harlow, D. Lane, Antibodies: A Laboratory Manu
al, Cold Spring Harbor Laboratory Press, (1988)).
Cells are labeled with ³⁵ S-cysteine for 8 hours 2 days after transfection. Culture medium was then harvested and cells were washed with detergent (RIPA buffer (150 mM NaCl, 1% NP-4.
0, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris (pH
7.5))) (Wilson, I. et al., Ibid. 37: 767 (1984)).
Both cell lysates and culture media are precipitated with HA-specific monoclonal antibodies. SD for precipitated proteins
Analyze by S-PAGE.

Example 3 Cloning of Ckβ-13 using the baculovirus expression system
DNA sequences encoding Ckβ-13 protein grayed and expressing full length (ATCC Accession No. 97113) is amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene: 5 'primer, And a BamHI restriction enzyme site (in bold), followed by 6 nucleotides (Kozak, M., J. Mol. Biol., 196, similar to a signal efficient for initiation of translation in eukaryotic cells).
947-950, (1987)), and the first 18 nucleotides of the Ckβ-13 gene (translation initiation codon "ATG" is underlined). The 3'primer is And contains a cleavage site for the restriction endonuclease Asp718, and 18 nucleotides complementary to the 3'untranslated sequence of the Ckβ-13 gene. Amplify the sequence using a commercially available kit (`` Geneclea
n "BIO 101 Inc., La Jolla, Ca.) is used to isolate from a 1% agarose gel. The fragment is then digested with the endonucleases BamHI and Asp718 and again purified on a 1% agarose gel. This fragment is called F2.

Vector pRG1 (mutant of pVL941 vector,
The following is used for the expression of Ckβ-13 protein using the baculovirus expression system (for a review, Summers, M. et al.
D. and Smith, GE. 1987, A manual of methods for
baculovirus vectors and insect cell culture proced
ures, Texas Agricultural Experimental Station Bull
etin No. (See 1555). This expression vector is Au
It contains the strong polyhedrin promoter of tographa californica nuclear polyhedrosis virus (AcMNPV), followed by recognition sites for the restriction endonucleases BamHI and Asp718. The simian virus (SV) 40 polyadenylation site is used for efficient polyadenylation. To facilitate selection of recombinant viruses, E. Insert the β-galactosidase gene from E. coli in the same direction as the polyhedrin promoter,
This is followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequence is flanked at both ends by the viral sequence for cell-mediated homologous recombination of cotransfected wild type viral DNA. Many other baculovirus vectors can be used in place of pRG1. For example, pA
c373, pVL941, and pAcIM1 (Luckow, VA and Summers, MD, Virology, 170: 31-39).

Plasmid restriction enzymes BamHI and Asp718
Digested with and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc. La Jolla, CA). This vector DNA is called V2.

Fragment F2 and dephosphorylated plasmid V2 are ligated with T4 DNA ligase. Then
E. A plasmid carrying the Ckβ-13 gene transformed into E. coli HB101 cells and using the enzymes BamHI and Asp718
Bacteria containing (pBac-Ckβ-13) are identified. The sequence of the cloned fragment is confirmed by DNA sequencing.

5 μg of the plasmid pBac-Ckβ-13 was treated with the lipofection method (Felgner et al. Proc. Natl. Acad. Sci. U).
SA, 84: 7413-7417 (1987)), using 1.0 μg of commercially available linearized baculovirus (“BaculoGold ^™ baculovirus”).
DNA ", Pharmingen, San Diego, CA.).

1 μg BaculoGold ^™ viral DNA and 5
μg of plasmid pBac-Ckβ-13 was added to 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, M
Mix in sterile wells of a microtiter plate containing D). Then 10 μl Lipofectin plus 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. The transfection mixture was then seeded with Sf9 insect cells (ATCC CRL 1) in 35 mm tissue culture plates with 1 ml Grace's medium without serum.
711) and add it dropwise. The plate is shaken back and forth to mix the newly added solution. The plates are then incubated at 27 ° C for 5 hours. 5 hours later
The transfection solution is removed from the plate and 1 ml Grace's insect medium supplemented with 10% fetal bovine serum is added. The plate is returned to the incubator and the culture is continued at 27 ° C for 4 days.

After 4 days, the supernatant is collected and the plaque assay is performed as described by Summers and Smith (supra). A modified method is the "Blue Gal" (Life Technologies), which allows easy isolation of blue-stained plaques.
Inc., Gaithersburg) is used. (A detailed description of the "plaque assay" can also be found in Life
User Guide for Insect Cell Culture and Baculovirology distributed by Technologies Inc., Gaithersburg, USA
(Pages 9-10).

After 4 days of serial dilution, virus is added to the cells and blue-stained plaques are picked with an Eppendorf pipette tip. The agar containing the recombinant virus is then resuspended in an Eppendorf tube containing 200 μl Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. After 4 days, the supernatants of these culture dishes were collected,
Then store at 4 ° C.

Sf9 cells were loaded with 10% heat-inactivated FBS-supplemented cells.
Grow in race medium. Multiplicity of infection (MOI)
2. Infect with recombinant baculovirus V-Ckβ-13 at 2.
After 6 hours, the medium was removed and methionine and
SF900 II medium (Life Technologies I
nc., Gaithersburg). 42 hours later 5 μCi
³⁵Of S-methionine and 5 μCi³⁵S cysteine (Amersh
am) is added. Incubate cells for an additional 16 hours
Cells and then harvested by centrifugation and labeled.
The recovered proteins by SDS-PAGE and autoradiography
To visualize.

Example 4 Expression Through Gene Therapy Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue was placed in tissue culture medium and divided into small pieces. A small section of tissue is placed on the moist surface of a tissue culture flask, with approximately 10 sections in each flask. Rotate the flask up and down, seal tightly, and leave at room temperature overnight. After 24 hours at room temperature, the flask is inverted and the tissue mass is kept fixed at the bottom of the flask and fresh medium (eg Ham's F12 medium with 10% FBS, penicillin, streptomycin) is added. Then
This is incubated at 37 ° C for about 1 week. At this time,
Fresh medium is added and replaced every few days thereafter. After an additional 2 weeks in culture, a monolayer of fibroblasts develops. The monolayer is trypsinized and scaled into a larger flask.

PMV-7 flanks the long terminal repeats of Moloney murine sarcoma virus (Kirschmeier, PT et al., DNA, 7: 219).
-25 (1988)) is digested with EcoRI and HindIII, followed by treatment with calf intestinal phosphatase. The linear vector is separated on an agarose gel and purified using glass beads.

CDNA encoding the polypeptide of the present invention
Are amplified with PRC primers corresponding to the 5'and 3'terminal sequences, respectively. The 5'primer contains an EcoRI site and the 3'primer further contains a HindIII site. Equal amounts of Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragments
Add together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for ligation of the two fragments. The ligation mixture is used to transform bacterial HB101, which is then plated on kanamycin-containing agar plates for the purpose of ensuring that the vector has the gene of interest inserted properly.

Amphoteric pA317 or GP + am12 packaging cells were treated with Dulbecco's Modified Eagle Medium (DME) containing 10% calf serum (CS), penicillin and streptomycin.
Grow to confluent density in tissue culture in M). The MSV vector containing the gene is then added to the medium and the packaging cells are transduced with the vector. At this point the packaging cells produce infectious viral particles containing the gene (at which point the packaging cells are called producer cells).

Fresh medium is added to the transduced producer cells, after which the medium is harvested from a 10 cm plate of confluent producer cells. Spent media containing infectious viral particles is filtered through a millipore filter to remove detached producer cells and then used to infect fibroblasts. The medium is removed from the semi-confluent plates of fibroblasts and quickly replaced with medium from producer cells. This medium is removed and replaced with fresh medium. If the titer of virus is high, virtually all fibroblasts will be infected and no selection is necessary. If the titer is very low, it is necessary to use a retroviral vector with a selectable marker (eg neo , or his ).

The engineered fibroblasts were then isolated,
Alternatively, they are injected into the host either after being grown to confluence on Cytodex 3 microcarrier beads. The fibroblast then produces the protein product.

Many modifications and variations of the present invention are possible in light of the above teachings, therefore, unless otherwise indicated, the present invention can be practiced within the scope of the appended claims.

[0155]

[Sequence list]

[Brief description of drawings]

FIG. 1 shows the cDNA sequence and the corresponding Ckβ-13.
The deduced amino acid sequence of The first 28 amino acids represent a leader sequence such that the putative mature polypeptide contains 65 amino acids. Use the standard one-letter abbreviations for amino acids. Sequencing is performed with 373 automated DNA sequencers (App
lied Biosysytems, Inc.).

FIG. 2 shows amino acid sequence homology between Ckβ-13 (top) and human MIP-1α polypeptide (bottom).

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 9/00 A61P 11/00 4C084 9/10 101 17/02 4H045 11/00 17/06 17/02 29/00 17 / 06 101 29/00 29/02 101 31/04 29/02 33/00 31/04 35/00 33/00 35/02 35/00 37/08 35/02 43/00 105 37/08 111 43 / 00 105 C07K 14/52 111 16/24 C07K 14/52 C12N 1/15 16/24 1/19 C12N 1/15 1/21 1/19 C12P 21/02 K 1/21 C12Q 1/02 5/10 1 / 68 A C12P 21/02 G01N 33/15 Z C12Q 1/02 33/50 Z 1/68 C12P 21/08 G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 A // C12P 21/08 A61K 37/02 (71) Applicant 597018381 9410 Key West Avenue, Rockville, Maryland 20850, United States of America (71) Applicant 591002957 Smithkline Beecham Corporation SMITHKLINE BEECHAM CORPORATION United States Pennsylvania 19406-0939, King of Prussia, Swedland Road 709 (72) Inventor Rehaodon United States Maryland 20878, Gaithersburg, Rutledge Drive 11033 (72) Inventor Seibel George United States Pennsylvania 19087, Saint Davids, Cornwall Circle 11F Term (reference) 2G045 AA40 4B024 AA01 AA11 BA21 CA04 CA09 DA02 DA06 EA02 EA04 GA11 GA18 GA19 HA03 HA14 4B063 QA01 QA18 QA19 QQ03 QQ20 QR77 QR55 QR32 QR32 QR32 QR32 QR32 QR32 QR32 QR32 QR32 QR32 QS34 QS39 QX01 QX07 4B064 AG02 AG27 CA02 CA10 CA19 CC24 CE02 CE06 CE12 DA01 DA13 4B065 AA26X AA90X AA93Y AB01 AC14 BA02 CA24 CA44 CA46 4C084 AA02 AA03 AA07 AA MAB MA2 MA66 MA2 MA14 MA2 MA14 MA16 MA16 MA59 MA63 MA63 ZB152 ZB212 ZB262 ZB272 ZB322 ZB372 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA01 DA76 DA86 EA22 EA23 EA28 EA50 FA72 FA74 GA15 GA26

Claims (20)

[Claims] 1. An isolated polynucleotide, which comprises:
The following: (a) a polynucleotide encoding a polypeptide containing amino acid-28 to amino acid 65 shown in SEQ ID NO: 2; (b) a polynucleotide encoding a polypeptide containing amino acid 1 to amino acid 65 shown in SEQ ID NO: 2. (C) a polynucleotide capable of hybridizing with said polynucleotide (a) or (b) and having at least 70% identity thereto; and (d) said polynucleotide (a), (b) or (c). An isolated polynucleotide comprising a member selected from the group consisting of: 2. The polynucleotide of claim 1, wherein the polynucleotide is DNA. 3. The polynucleotide of claim 2 which encodes a polypeptide comprising amino acids -28 to 65 of SEQ ID NO: 2. 4. The polynucleotide of claim 2 which encodes a polypeptide comprising amino acids 1-65 of SEQ ID NO: 2. 5. An isolated polynucleotide, which comprises:
The following: (a) a polynucleotide encoding a polypeptide having an amino acid sequence expressed by the DNA contained in ATCC Deposit No. 97113; (b) capable of hybridizing with the polynucleotide (a), and at least 70% thereof. An isolated polynucleotide comprising a member selected from the group consisting of a polynucleotide that is identical to: (c) the polynucleotide fragment of polynucleotide (a) or (b). 6. A vector comprising the DNA of claim 2. 7. A host cell genetically engineered with the vector of claim 6. 8. From the host cell of claim 7, the DN
A method of producing a polypeptide comprising the step of expressing a polypeptide encoded by A. 9. A method for producing a cell capable of expressing a polypeptide, which comprises the step of genetically engineering the cell with the vector according to claim 6. 10. A polypeptide, which is (i) SEQ ID NO: 2.
A polypeptide having the deduced amino acid sequence of, and fragments, analogs, and derivatives thereof;
A polypeptide selected by the cDNA of ATCC Deposit No. 97113, and a polypeptide selected from the group consisting of fragments, analogs, and derivatives of the polypeptide. 11. A compound that mimics the activity of the polypeptide of claim 10. 12. A compound that antagonizes the activity of the polypeptide of claim 10. 13. An antibody against the polypeptide of claim 10, comprising a member selected from the group consisting of monoclonal and polyclonal antibodies. 14. A method for treating a patient in need of Ckβ-13, wherein the patient is in a therapeutically effective amount.
Administering the polypeptide of claim 1. 15. The method of claim 13, wherein the therapeutically effective amount of the polypeptide is administered by providing a patient with DNA encoding the polypeptide and expressing the polypeptide in vivo. . 16. A method of treating a patient in need of inhibiting a Ckβ-13 polypeptide comprising: administering to the patient a therapeutically effective amount of a compound of claim 12. . 17. A method of diagnosing a disease or susceptibility to underexpression of the polypeptide of claim 10 comprising the step of detecting a mutation in the nucleic acid sequence encoding the polypeptide. Method. 18. A diagnostic method comprising the step of analyzing the presence of the polypeptide of claim 10 in a sample derived from a host. 19. A method for identifying a compound that is active as an agonist to the polypeptide of claim 10, wherein: (a) the cell normally migrates in response to the polypeptide of claim 13. Mixing the compound to be screened with a reaction mixture containing the cells under conditions that: and, if the compound is effective as an agonist, determining the extent of migration of the cells for identification. The method of including. 20. The method of claim 18, wherein the Ckβ-13 polypeptide is added in the mixture of step (a) and the determination of the extent of migration identifies compounds that are effective as antagonists.