MXPA97009192A - Protein 4 chemotherapy of monoci - Google Patents
Protein 4 chemotherapy of monociInfo
- Publication number
- MXPA97009192A MXPA97009192A MXPA/A/1997/009192A MX9709192A MXPA97009192A MX PA97009192 A MXPA97009192 A MX PA97009192A MX 9709192 A MX9709192 A MX 9709192A MX PA97009192 A MXPA97009192 A MX PA97009192A
- Authority
- MX
- Mexico
- Prior art keywords
- polypeptide
- cells
- mcp
- dna
- host
- Prior art date
Links
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Abstract
A polypeptide of human monocyte chemoattractant protein 4, and DNA (RNA) coding for such a polypeptide, and a method for the production of such polypeptide by recombinant techniques are described. They also describe methods for the use of such a polypeptide to prevent and / or treat the mobilization of totipotential cells, myeloprotection and neuronal protection, to treat tumors, to promote wound healing, to combine parasitic infection and to regulate the hematopoiesis. Antagonists against such a polypeptide are also described, which can be used to treat rheumatoid arthritis, pulmonary inflammation, allergy infectious diseases and to prevent inflammation and atherosclerosis. Diagnostic assays are also described to identify mutations in the nucleic acid sequence encoding a polypeptide of the invention, and to detect altered levels of the polypeptide of the present invention, to detect disease
Description
PROTEIN 4 CHEMOTHERAPY OF MONOCYTES
DESCRIPTION OF THE INVENTION
This invention relates to the newly identified polynucleotides, to the polypeptides encoded by such polynucleotides, to the use of such polynucleotides and polypeptides, as well as to the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is the monocyte chemotactic protein 4 (MCP-4). The invention also relates to the inhibition of the action of such polypeptides. There are three forms of monocyte chemotactic protein, namely, MCP-1, MCP-2 and MCP-3. All these proteins have been structurally and functionally characterized and have also been cloned and expressed. MCP-1 and MCP-2 have the ability to attract leukocytes (monocytes and leukocytes), while MCP-3 also attracts eosinophils and T lymphocytes (Dahinderi, E. et al., J. Exp. Med. 179: 751-756 ( 1994)). Initially, the specific attractant factor of human monocytes was purified from a glioma cell line and a cell line from
REF: 26079
monocytes. Matsushima, K. and collaborators, J. Exp. Med. 169: 1485-1490 (1989). This factor was originally designated glioma-derived chemotactic factor (GDCF) and monocyte activation and chemotactic factor (MCAF) by Matsushima et al. This factor is now referred to as MCP-1. Subsequent cloning of the cDNA for MCP-1 showed that it is highly similar to the murine JE gene. The JE gene could be massively induced in murine fibroblasts by platelet-derived growth factor. Cochran, B.H. et al., Cell 33: 939-947 (1983). Murine JE is highly similar to MCP-1. The MCP-1 protein has 62% identity to the murine JE in a region of 68 shared N-terminal residues. It is widely accepted that JE and MCP-1 are homologous species. A method for suppressing tumor formation in a vertebrate by administration of JE-MCP-1 has been described in PCT application WO-92/20372, together with methods for the treatment of localized complications of malignancies, and methods to combat parasitic infection by administering JE / MCP-1. It was found that the expression of JE / MCP-1 protein in cells
malignant, suppresses the ability of cells to form tumors in vi vo. Human MCP-1 is a basic peptide of 76 amino acids with a predicted molecular mass of 8,700 daltons. MCP-1 is inducibly expressed mainly in monocytes, endothelial cells and fibroblasts. Leonard, E.J. and Yoshimura, T., Immunol. Today 11: 97-101 (1990). The factors that induce this expression are Interleukin 1, TNF or treatment with lipopolysaccharide. Other properties of MCP-1 include the ability to strongly activate mature human basophils in a manner sensitive to pertussis toxin. MCP-1 is a cytokine capable of directly inducing the release of histamine by basophils (Bischoff, S.C. et al., J. Exp. Med. 175: 1271-1275 (1992)). In addition, MCP-1 promotes the formation of leukotriene C4 by basophils pre-treated with Interleukin 3, Interleukin 5, or granulocyte / acrophage colony stimulation factor. The release of the mediator of basophils induced by MCP-1 may play an important role in allergic inflammation and in other pathologies that express MCP-1.
Clones having a nucleotide sequence encoding a human monocyte activating chemotactic factor (MCAF) reveal the primary structure of the MCAF polypeptide which is composed of a putative signal peptide sequence of 23 amino acid residues and a mature MCAF sequence of 76 amino acid residues. Furutani, Y.H., and collaborators, Bi ochem. Bi ophys. Res. Commu. 159: 249-55 (1989). The complete amino acid sequence of the monocyte chemotactic factor derived from human glioma (GDCF-2) has also been determined. This peptide attracts human monocytes but not neutrophils. It was established that GDCF-2 comprises 76 amino acid residues. The peptide chain contains 4 hemicysteins, in positions 11, 12, 36 and 52, which create a pair of curls, accumulated in disulfide bridges. In addition, the MCP-1 gene has been designated to human chromosome 17. Mehrabian, M.R., and collaborators, genomi cs 9: 200-3 (1991). Some data suggest that a potential role for MCP-1 is mediating the infiltration of monocytes from the arterial wall. Monocytes appear to be central or important for atherogenesis, either as progenitors of the foam cells and
as a potential source of growth factors that mediate intimal hyperplasia. Nel en, N.A., et al., J. Clin. Inves t. 88: 1121-7 (1991). It has also been found that synovial production of MCP-1 may play an important role in the recruitment of mononuclear phagocytes during the inflammation associated with rheumatoid arthritis, and that synovial tissue macrophages are the dominant source of this cytokine. It was found that the levels of MCP-1 are significantly higher in synovial fluid from patients with rheumatoid arthritis compared to synovial fluid from patients with osteoarthritis or patients with other arthritis. Koch, A.E., and collaborators, J. Cl in. Inves t. 90: 772-9 (1992). MCP-2 and MCP-3 are classified in a subfamily of proinflammatory proteins and are functionally related to MCP-1 because they specifically attract monocytes, but not neutrophils. Van Damme, J., et al., J. Exp. Med. 176: 59-65 (1992). MCP-3 shows 71% and 58% amino acid homology to MCP-1 and MCP-2 respectively. MCP-3 is an inflammatory cytokine that regulates the functions of macrophages.
Transplantation of hemolymphopoietic totipotential cells has been proposed in the treatment of cancer and hematological disorders. Many studies show that transplantation of haematopoietic cells, harvested from the peripheral blood, has advantages over the transplantation of totipotential cells derived from the bone marrow. Due to the lower number of circulating totipotential cells, there is a need for the induction of mobilization of cells from the pluripotential or totipotential line of the bone marrow to the peripheral blood. The reduction of the amount of blood to be processed to obtain an adequate amount of totipotent cells could increase the use of the autotransplant procedures and eliminate the risk of the graft versus host reaction related to allograft. Currently, the blood mobilization of cells of the CD34 'line of bone marrow is obtained by the injection of a combination of agents, including antiblastic drugs and G-CSF or GM-CSF. Drugs that are capable of mobilizing totipotent cells include IL-1, IL-7, IL-8, and NIP-la. IL-1 and IL-8 demonstrate proinflammatory activity that can be dangerous for
the good graft. IL-7 must be administered at high doses for a prolonged duration and MlP-la is not very active as a simple agent, and shows better activity when combined with G-CSF. In accordance with one aspect of the present invention, a new mature polypeptide is provided, as well as diagnostically or therapeutically useful biologically active fragments, analogs and derivatives thereof. The polypeptide of the present invention is of human origin. According to yet another aspect of the present invention, isolated nucleic acid molecules are provided that encode a polypeptide of the present invention, including mRNAs, DNAs, cDNAs, genomic DNAs as well as diagnostically or therapeutically useful biologically active analogs and fragments. thereof. According to yet another aspect of the present invention, there is provided a process for the production of such a polypeptide by recombinant techniques comprising the culture of prokaryotic and / or eukaryotic recombinant host cells, which contain a nucleic acid sequence coding for a polypeptide of the present invention,
under conditions that promote the expression of the protein and the subsequent recovery of the protein. According to a further aspect of the present invention, there is provided a process for using such a polypeptide, or the polynucleotide encoding such a polypeptide for therapeutic purposes, for example, for the mobilization of totipotential cells., for myeloprotection, and neuronal protection, to treat tumors, to promote wound healing, to combat parasitic infection and to regulate hematopoiesis. In accordance with a further aspect of the present invention, antibodies against such polypeptides are provided. According to yet another aspect of the present invention, agonists that mimic the polypeptide of the present invention are provided, and bind to the receptors to elicit responses from the second messenger. According to yet another aspect of the present invention, antagonists are provided for such polypeptides, which can be used to inhibit the action of such polypeptides, for example, in the treatment of rheumatoid arthritis, pulmonary inflammation, allergy,
infectious diseases and to prevent inflammation and atherosclerosis. In accordance with a further aspect of the present invention, nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention are also provided. According to yet another aspect of the present invention, diagnostic assays are provided for detecting diseases or susceptibility to diseases related to mutations in the nucleic acid sequences encoding a polypeptide of the present invention. According to a further aspect of the present invention, there is provided a process for using such polypeptides, or the polynucleotides encoding such polypeptides, for purposes related to scientific research, for example, DNA synthesis and manufacturing. of DNA vectors. These and other aspects of the present invention should be apparent to those
experts in the art from the teachings herein. The following drawings are illustrative of the embodiments of the invention, and are not intended to limit the scope of the invention, as encompassed by the claims.
Figure 1 describes the cDNA sequence and the corresponding deduced amino acid sequence of MCP-4. The sequence of 119 amino acids shown is the full-length protein, with approximately the first 26 amino acids representing a leader sequence (underlined) such that the mature form of the protein is 93 amino acids in length. The standard one-letter abbreviation for amino acids is used.
Figure 2 illustrates a comparison of the homology of the amino acid sequence between the polypeptide of the present invention, MCP-1 and MlP-la.
MCP-4 shows 39% homology with MlP-la and 34% homology with MCP-1.
Figure 3 illustrates the chemotactic activity of the polypeptide of the present invention
on neutrophils (PMN) and peripheral blood mononuclear cells (PBMC). Neutrophils and peripheral blood mononuclear cells were isolated from peripheral blood, loaded with calcein-AM and used for chemotaxis in a single-use, 96-well Neuroprobe chemotherapy chamber. After 90 minutes of incubation with MCP-4, the chamber was dismounted, the filter was air-dried and the number of cells that migrated through the membrane were quantified in a Cytofluor II.
Figure 4 illustrates that MCP-4 inhibits the growth and differentiation of potential, highly proliferative colony forming cells (HPP-CPC) (A) and is not effective on low proliferation potential colony forming cells (LPP-CFC ) (B). In these experiments, 1,500 cells from non-adherent, low-density bone marrow cells were plated on agar medium supplemented with 5 ng / ml mouse IL-3, 100 ng / ml mouse SCF, ng / ml of mouse IL-la, 5 ng / ml of human M-CSF, and with or without the indicated concentrations of MCP-4. The colonies were evaluated after 14
days of incubation. Three experiments were performed. The results are presented as the mean number of colonies ± the standard deviation (SD). An irrelevant protein had no effect.
Figure 5 shows the effect of MCP-4 on bone marrow cells, which were enriched in the primitive Lin lines by eliminating the committed precursor cells (anti-CDII, CD4, CD8, CD45R and Gr-1 antibodies). ). Panel A shows the ± SD ratios of LPP-CFC / HPP-CFC in bone marrow cells (column 1) or Lin cells (column 2) plated on agar medium with 5 ng / ml of IL- 3, 100 ng / ml SCF, 10 ng / ml IL-la, 5 ng / ml M-CSF. Columns 3, 4 and
show the proportion of LPP-CFC / HPP-CFC found in Lin cells that were cultured with 5 ng / ml of IL-3 and 100 ng / ml of SCF (column 3), IL-3, SCF and 50 ng / ml of MCP-4 (column 4) or IL-3, SCF and 50 ng / ml of an irrelevant protein (column 5). After 6 days, the cultures were evaluated for HPP-CFC and LPP-CFC. Panel B shows cellularity after
6 days of incubation.
Figure 6 illustrates that MCP-4 protects HPP-CFC but not LPP-CFC from the cytotoxic effect of cytosine arabinoside (Ara-C) in vi tro.
Figure 7 illustrates that MCP-4 protects HPP-CPC but not LPP-CFC from the cytotoxic effect of 5-fluorouracil (5-FU) in vi tro.
Figure 8 illustrates the effect of MCP-4 and basic FGF on Cortical Neuronal Survival.
In accordance with one aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID No. 2) or for the mature encoded polypeptide by the cDNA of the clone deposited as ATCC Deposit No. 75703 on March 10, 1994. The polynucleotide of this invention was described from a genomic library of activated monocyte cDNAs. It contains an open reading frame that codes for a protein of approximately 119 amino acids in length, of which the first 26 amino acid residues comprise a putative leader sequence. It is predicted
therefore, the mature protein is 93 amino acids in length. This is structurally related to the mouse monocyte chemotactic protein (MCP-1 or JE), showing 27% identity, and 56% similarity over the complete, human MCP-1 protein sequence. The polypeptide contains the four cysteine residues that appear in all chemokines in a characteristic portion. The spacing between these cysteines is conserved compared to murine MCP-1 / JE, strongly suggesting that the new gene is a chemokine. The polynucleotide of the present invention can be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA can be double-stranded or single-stranded, and if it is single-stranded it can be the coding strand or the non-coding strand (antisense). The coding sequence coding for the mature polypeptide can be identical to the coding sequence shown in Figure 1 (SEQ ID No. 1) or that of the deposited clone, or it can be a different coding sequence whose coding sequence, as result of the redundancy or degeneracy of the genetic code,
encodes for the same mature polypeptide as the DNA of Figure 1 (SEQ ID No. 1) or the deposited cDNA. The polynucleotide encoding the mature polypeptide of Figure 1 (SEQ ID No. 1) or for the mature polypeptide encoded by the deposited cDNA may include, but is not limited to: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and the additional coding sequence such as a guiding or secretory sequence, or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally the additional coding sequence) and the non-coding sequence, such as the introns or the 5 'and / or 3' sequence of non-coding coding sequence for the mature polypeptide . Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide that includes only the coding sequence for the polypeptide, as well as a polynucleotide that includes the additional coding and / or non-coding sequence. The present invention also relates to the variants of the polynucleotides described above.
described herein, which encode fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID No. 2) or the polypeptide encoded by the cDNA of the deposited clone. The variant of the polynucleotide can be an allelic variant of natural origin of the polynucleotide or a non-natural variant of the polynucleotide. Thus, the present invention includes polynucleotides that encode the same mature polypeptide shown in Figure 1 (SEQ ID No. 2) or the same mature polypeptide encoded by the cDNA of the deposited clone., as well as variants of such polynucleotides, whose variants encode 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. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants. As indicated hereinabove, the polynucleotide may have a coding sequence which e-s is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SEQ ID No. 1) or the sequence of
coding of the deposited clone. As is known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide. The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide can be fused in the reading frame for the polynucleotide sequence that aids the expression and secretion of a polypeptide from a host cell, e.g. , a guiding sequence that functions as a secretory sequence to control the transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell, to form the mature form of the polypeptide. The polynucleotides can also encode a proprotein which is the mature protein plus additional 5 'amino acid residues. A mature protein that has a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved, an active mature protein remains. In this way,
example, the polynucleotide of the present invention can encode a mature protein, or for a protein having a prosequence, or for a protein having a prosequence or for a protein having a prosequence and a presequence (leader sequence). The polynucleotides of the present invention may also have the coding sequence fused in the structure to a marker sequence that allows purification of the polypeptide of the present invention. The marker sequence may be a hexahistidine tag supplied by a pQE-9 vector to provide for the purification of the mature polypeptide fused to the tag, in the case of a bacterial host, or, for example, the tag sequence may be a hemagglutinin tag ( HA) when a mammalian host is used, for example COS-7 cells. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I. et al., Cell 37: 767 (1984)). The term "gene" means the segment of DNA involved in the production of a polypeptide chain; this includes the preceding and subsequent regions to the coding region (forward and
back) as well as the intervention sequences (introns) between the individual coding segments (exons). Fragments of the full-length gene of the present invention can be used as a hybridization probe for a cDNA genomic library, to isolate the full-length cDNA and to isolate other cDNAs, which have high sequence similarity to the gene, or similar to biological activity. Probes of this type preferably have at least 30 bases, and may contain, for example, 50 or more bases. The probe can also be used to identify a cDNA clone corresponding to a full-length transcript and a clone or genomic clones containing the complete gene that includes the regulatory and promoter regions, exons and introns. An example of a selection comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. The labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to select a human cDNA library, DNA
genomic or mRNA, to determine which members of the library the probe hybridizes to. The present invention also relates to polynucleotides that hybridize to the sequences previously described herein, if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention relates particularly to polynucleotides that hybridize under stringent conditions to the polynucleotides described hereinabove. As used in this, the term "stringent conditions" means that the hybridization will occur only if there is at least 95%, and preferably at least 97% identity between the sequences. Polynucleotides that hybridize to the polynucleotides described above in a preferred embodiment code for polypeptides that retain either substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID No. 1) or the deposited cDNAs. Alternatively, the polynucleotide can have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases, which hybridize to a polynucleotide of the present invention,
and which has an identity to it, as described hereinabove, and which may or may not retain its activity. For example, such polynucleotides can be used as probes for the polynucleotide of SEQ ID No. 1, for example, for the recovery of the polynucleotide or as a diagnostic probe or as a PCR primer. Thus, the present invention is directed to polynucleotides having at least an identity of 70%, preferably at least 90%, and more preferably at least 95% identity to a polynucleotide encoding the polypeptide of SEQ ID No. 2, as well as fragments thereof, the fragments of which have at least 30 bases and preferably at least 50 bases, and the polypeptides encoded by such polynucleotides. The deposit (s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art, and are not an admission that a deposit under 35 U.S.C. section 112. The sequence of polynucleotides
contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded by these, are incorporated herein by reference, and are of control in the case of any conflict with any description or sequences herein. A license to manufacture, use or sell the deposited materials may be required, and no such license is hereby granted. The present invention further relates to a polypeptide which has the deduced amino acid sequence of Figure 1 (SEQ ID No. 2) or which has the amino acid sequence encoded by the deposited cDNA, as well as the fragments, analogs and derivatives of such a polypeptide. The terms "fragment", "derivative" and "analogue" when referring to the polypeptide of Figure 1 (SEQ ID No. 2) or that encoded by the deposited cDNA, means a polypeptide that retains essentially the same biological function or the activity as such polypeptide. Thus, an analog includes a proprotein that can be activated by cleaving the proprotein portion to produce an active mature polypeptide.
The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide. The fragment, derivative or analogue of the polypeptide of Figure 1 (SEQ ID No. 2) or that encoded by the deposited cDNA can be i) one in which one or more of the amino acid residues are substituted with a conserved amino acid residue or non-conserved (preferably a conserved amino acid residue), and such a substituted amino acid residue may or may not be one encoded by the genetic code, or ii) one in which one or more of the amino acid residues includes a substituent group, or iii) one in which the mature polypeptide is fused to another compound, such as a compound to increase the half-life of the polypeptide (e.g., polyethylene glycol), or iv) one in which the additional amino acids are fused to the mature polypeptide, such as a guiding sequence or secretory sequence, or a sequence that is employed for the purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are considered to be within the scope of
those skilled in the art, from the teachings herein. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and are preferably purified to homogeneity. The term "isolated" means that the material is removed from its original environment (for example, the natural environment if it is of natural origin). For example, a polynucleotide of natural origin or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the materials coexisting in the natural system, is isolated. Such polynucleotides could be part of a vector and / or such polynucleotides c polypeptides could be part of a composition, and still be isolated, since such a vector or composition is not part of their natural environment. The polypeptides of the present invention include the polypeptide of SEQ ID No. 2 (in particular the mature polypeptide) as well as the polypeptides having at least 70% similarity
(preferably at least 70% identity) to the polypeptide of SEQ ID No. 2, and more preferably
at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID No. 2, and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID No. 2, and also include portions of such polypeptides with such a portion of the polypeptide that generally contains at least 30 amino acids, and more preferably at least 50 amino acids. As is known in the art "similarity" between two polypeptides is determined by comparison of the amino acid sequence and its conserved amino acid substitutes of a polypeptide to the sequence of a second polypeptide. The fragments or portions of the polypeptides of the present invention can be used for the production of the corresponding full length polypeptide, by peptide synthesis; therefore, the fragments can be used as intermediates for the production of full-length polypeptides. Fragments or portions of the polynucleotides of the present invention can be used to synthesize the full length polynucleotides of the present invention.
The present invention also relates to vectors that include polynucleotides of the present invention, host cells that are engineered with vectors of the invention, and the production of polypeptides of the invention by recombinant techniques. The host cells are engineered (transduced or transformed or transfected) with the vectors of this invention, which may be, for example, a cloning vector or an expression vector. The vector can be, for example, in the form of a plasmid, a viral particle, a phage, etc. Genetically engineered host cells can be cultured in modified conventional nutrient media as appropriate to activate the promoters, select transformants or amplify the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to one of ordinary skill in the art. The polynucleotides of the present invention can be used to produce polypeptides by recombinant techniques. In this way,
example, the polynucleotide can be included in any of a variety of expression vectors for the expression of a polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, for example, derived from SV40; bacterial plasmids; Phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia virus, adenovirus, fowlpox virus, and pseudorabies. However, any other vector can be used, as long as it is replicable and viable in the host. The appropriate DNA sequence can be inserted into the vector by a variety of methods. In general, the DNA sequence is inserted into one or several appropriate restriction endonuclease sites, by methods known in the art. Such procedures and others are considered to be within the scope of those of skill in the art. The DNA sequence in the expression vector is operatively linked to one or more expression control sequences, appropriate (promoters) to direct the synthesis of mRNA. As representative examples of such promoters,
they can mention: promoter LTR or SV40, lac or trp of E. coli, PL promoter of phage lambda and other promoters that are known to control the expression of genes in prokaryotic or eukaryotic cells, or their viruses. The expression vector also contains a ribosome binding site, for the start of translation and a transcription terminator. The vector may also include the appropriate sequences for the amplification of expression. In addition, the expression vectors preferably comprise one or more selectable marker genes to provide a phenotypic trait for the selection of transformed host cells, such as dihydrofolate reductase or resistance to neomycin for the culture of eukaryotic cells, or as resistance to tetracycline or ampicillin in E. coli. The vector containing the appropriate DNA sequence as described hereinabove, as well as an appropriate promoter or control sequence, can be employed to transform an appropriate host, to allow the host to express the protein. As representative examples of the appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces,
Salmonel l a typhimuri um; fungal cells, such as yeast; insect cells such as Drosophil to S2 and Spodoptera Sf9; animal cells, such as CHO, COS or melanoma; adenovirus; plant cells, etc. The selection of an appropriate host is considered to be within the reach of those of experience in the art from the teachings herein. More particularly, the present invention also includes recombinant constructs that comprise one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, within which a sequence of the invention has been inserted, in a forward or inverse orientation. In a preferred aspect of this invention, the construct further comprises the regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of appropriate vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pDlO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene);
ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5
(Pharmacia); Eukaryotic; pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL
(Pharmacia). However, any other plasmid or vector can be used, as long as they are replicable and viable in the host. The promoter regions can be selected from any desired gene using the CAT vectors (chloramphenicol transferase) or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. The particular named bacterial promoters include lacl, lacZ, T3, T7, gpt, lambda PR PL and trp. Eukaryotic promoters include immediate early CMV, HSV thymidine kinase, early or early SV40 and SV40, retrovirus LTRs, and mouse metallothionein-I. The selection of the appropriate vector and promoter is very well within the level of skill in the art. In a further embodiment, the present invention relates to host cells containing the constructions described above. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a cell
yeast, or the host cell can be a prokaryotic cell, such as a bacterial cell. The introduction of the construct into the host cell can be effected by transfection with calcium phosphate, transfection mediated by DEAE-dextran, or electroporation (Davis, L., Dibner, M., Battey, I., Basi c Me thods in Molecular Bi olgy, (1986)). Constructs in the host cells can be used in a conventional manner to produce the genetic product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers. Mature proteins can be expressed in mammalian cells, in yeast, bacteria or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins, using RNAs derived from the DNA constructs of the present invention. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Mol ecul ar
Cloning: A Labora tory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the description of which is incorporated by reference herein. The transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by the insertion of an enhancer sequence into the vector. Augmentators are elements that act in cis position of DNA, usually approximately 10 to 300 base pairs acting on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin from 100 to 270 base pairs, an enhancer of the early cytomegalovirus promoter, the polyoma enhancer on the late side of the replication origin, and adenoviral enhancers. In general, recombinant expression vectors will include origins of replication and selectable markers that allow the transformation of the host cell, for example, the ampicillin resistance gene of the TRP1 gene of S. cerevi siae and E. coli, and a promoter derived from a highly expressed gene, to direct the transcription of a downstream structural sequence. Such promoters can be derived from
operons coding for glycolytic enzymes such as 3'-phosphoglycerate kinase (PGK), factor a, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in the appropriate phase with the translation initiation and termination sequences, and preferably, a guiding sequence capable of directing the secretion of the translated protein into the periplasmic space or into the extracellular medium. Optionally, the heterologous sequence can encode a fusion protein that includes an N-terminal identification peptide that imparts the desired characteristics, for example, stabilization or simplified purification of the expressed recombinant product. Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence coding for a desired protein, together with the translation initiation and termination signals, appropriate in operable reading phase with a functional promoter. The vector will comprise one or more selectable phenotypic markers, and an origin of replication to ensure vector maintenance, and if desirable, to provide amplification within the vector.
of the host. Suitable prokaryotic hosts for transformation include E. coli, Bacill us subtili s, Salmonell a typhimuri um and various species within the genera Pseudomonas, Streptomyces, and Staphyl ococcus, although others may also be employed, as a matter of choice. As a representative but not limiting example, expression vectors useful for bacterial use may comprise a selectable marker and the bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well-known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMÍ
(Promega Biotec, Madison, Wl, USA). These "backbone" sections of pBR322 are combined with an appropriate promoter and the structural sequence to be expressed. After transformation of an appropriate host strain and development of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature change or chemical induction) and the cells are cultured for an additional period. .
The cells are typically harvested by centrifugation, broken or destroyed by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells used in the expression of proteins can be destroyed or broken by any convenient means, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysis agents, such methods being known to those skilled in the art. The technique. Various mammalian cell culture systems can also be employed to express the recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, CeIJ 23: 175 (1981), and other cell lines capable of expressing a compatible vector, e.g. cellular C127, 3T3, CHO, HeLa and BHK. The mammalian expression vectors will comprise an origin of replication, an appropriate promoter and an enhancer, and also any necessary ribosome binding sites, the polyadenylation site, the donor and acceptor sites of splicing, the termination sequences of
the transcript, and the non-transcribed sequences flanking the 5 'end. The DNA sequences derived from the SV40 junction, and the polyadenylation sites can be used to provide the required non-transcribed genetic elements. The polypeptide can be recovered and purified from recombinant cell cultures by methods that include precipitation with ammonium sulfate or ethanol, acid extraction, anionic or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. The steps for protein refolding may be used, as necessary, to complete the configuration of the mature protein. Finally, high-performance liquid chromatography (HPLC) can be used for the final purification steps. The polypeptides of the present invention may be a naturally purified product, or a product of synthetic chemical processes, or produced by recombinant techniques from a prokaryotic or eukaryotic host (e.g.
by bacterial cells, yeast, higher plants, insect and mammal, in culture). Depending on the host employed in a recombinant production process, the polypeptides of the present invention may be glycosylated or may not be glycosylated. The polypeptides of the present invention may also include an initial methionine amino acid residue. The polypeptide of the present invention can be used for the promotion of wound healing. Since MCP-4 is a chemokine, it is an attractant for leukocytes (such as monocytes, T lymphocytes, basophils, PMNs, PBLs, etc.); therefore, it causes infiltration of target immune cells into the wound area. The MCP-4 polypeptide can also be used as an antitumor treatment and for the treatment of localized complications of a malignancy, such as pleural effusions or ascites. The infiltration of MCP-4 into the anatomical space involved can lead to the accumulation and activation of local monocytes. The presence of MCPs i n vi is accompanied by a local increase in the presence of eosinophils, which have the distinctive function
to kill the larvae of parasites that invade tissues, such as schistosomiasis, trichinosis and ascariasis. Therefore, MCP-4 can be used to combat parasitic infections. The polypeptide of the present invention can be used to mobilize the hematopoietic progenitor cells into the peripheral blood circulation of a human or non-human host, preferably a human host, for subsequent recovery and use thereof in transplantation. The polypeptide of the present invention is administered in an amount effective to mobilize inwards and increase the amount of hematopoietic progenitor cells in the peripheral blood, in particular, to implement the amount of human cells of the hematopoietic line in the peripheral blood. Such cells are frequently referred to as CD34 + cells. For example, the polypeptide is administered in amounts as described hereinafter. The polypeptide of the present invention can be administered alone or in conjunction with other agents, for example, GM-CSF and G-CSF, which are known to be effective in increasing such cells in the peripheral blood. The mobilization of progenitor cells
hematopoietic to the peripheral circulation is important for the autologous and heterologous bone marrow transfers that are used, for example, for the treatment of cancer and hematological disorders. The polypeptide of the present invention can also be employed to inhibit the destruction of progenitor-hematopoietic cells in a non-human and human host, preferably a human host, resulting from treatment with chemotherapeutic agents. The polypeptide of the present invention can be administered before, during or subsequent to chemotherapy, and allows a higher dose of chemotherapy to be employed in the treatment of cancer. The polypeptide of the present invention is administered in an amount effective to inhibit the destruction of the hematopoietic progenitor cells; for example, the polypeptide is administered in amounts as described hereinafter. The polypeptide can be administered alone or in conjunction with other agents. The polypeptide of the present invention can also be used to protect the hematopoietic progenitor cells, to prevent with this or to inhibit the diseases that can result from the
destruction of them; for example, leukopenia, myelodysplastic syndrome, and neutropenia. The polypeptide of the present invention can also be employed in effective amounts to inhibit the degradation of neuronal cells in human and non-human hosts, preferably a human host, which results from degenerative neuronal diseases such as Alzheimer's disease, of Parkinson's and the complex related to AIDS. For example, the polypeptide can be used in amounts as described hereinafter.
Table 1
Effect of administration of MCP-4 to mice on the distribution of primitive hematopoietic progenitors in peripheral blood, spleen and bone marrow after two days
Progenitor Numbers by Treatment 10 'PB cells 104 spleen cells 104 BM cells HPP LPP IM HPP LPP IM HPP LPP
PB = Peripheral blood, mononuclear cells Spl = Fraction of low density of splenic cells BM = Fraction of bone marrow that is 6 times enriched for primitive cells
HPP = highly proliferative and proliferating colony forming cells LPP = sparingly proliferating potential colony cells IM = immature cell, a rare type of cell found in the bone marrow, gives rise to a small highly collapsing colony (less than 50 5 cells / colony) in the presence of multiple cytokines; the cells within the colony are stacked in a horizontal plane and show nuclear staining characteristics similar to blasts.
Three mice were injected intraperitoneally daily with either MCP-
4 or saline solution. 48 hours after the first injection, blood was collected from each animal by cardiac puncture and the mice were sacrificed to obtain bone marrow and spleen. The indicated numbers of cells from each of the tissues were then plated in duplicate in medium containing agar, in the presence of rmIL-3 (5 ng / ml), rmSCF 15 (50 ng / ml), rhM-CSF (5 ng / ml), and rmlL-la (10 ng / ml) and incubated for 14 days. The data are combined from three animals in each group and expressed as the mean ± the standard deviation.
Table 2 Effect of administration of MCP-4 to mice on the distribution of primitive haematopoietic progenitors in peripheral blood, spleen and bone marrow after four days
Progenitor Numbers by Treatment 10; PB cells 10 * spleen cells 104 BM cells HPP LPP IM HPP LPP IM HPP LPP
PB = Peripheral blood, mononuclear cells Spl = Fraction of low density of splenic cells BM = Fraction of bone marrow that is 6 times enriched for primitive cells
HPP = Highly proliferating and potential colony forming cells LPP = Scarcely forming cells of prolific potential colonies IM = Immature cell, a rare type of cell found in the bone marrow, gives rise to a small highly retractable colony (less than 50 5 cells / colony) in the presence of multiple cytokines; the cells within the colony are stacked in a horizontal plane and show nuclear staining characteristics similar to blasts.
Three mice were injected intraperitoneally daily with either MCP-10 or saline. 48 hours after the first injection, blood was collected from each animal by cardiac puncture and the mice were sacrificed to obtain bone marrow and spleen. The indicated numbers of cells from each of the tissues were then plated in duplicate in medium containing agar, in the presence of rmIL-3 (5 ng / ml), rmSCF 15 (50 ng / ml), rhM-CSF (5 ng / ml), and rmlL-la (10 ng / ml) and incubated for 14 days. The data are combined from three animals in each group and expressed as the mean ± standard deviation
Table 3 Analysis of peripheral blood leukocyte composition by FACSan in mice administered with MCP-4 after two days
Percent positive in transferred cell populations Treatment CD45R + GR.l + Mac .1 + CD8 f CD4 + B cells PMN Monocytes T cells T cells
• b cp
Three C57 Black 6 mice (approximately 20 g in weight) were injected intraperitoneally daily with saline or MCP-4. 48 hours after the first injection, the blood was harvested by cardiac puncture and the mice were sacrificed to obtain spleen and bone marrow cells. For immunostaining, 0.1 ml of blood from each of the 10 animals was first treated with the Gen Trak lysis solution to break red blood cells. The nucleated cells were then pelleted,
washed with PBS, and incubated with monoclonal antibodies conjugated to PE, against CD45R, Gr.l, Mac.l, CD4, and CD8, and processed for flux ocitometry. At least 10,000 cells were analyzed. The data is expressed as the mean of the percent of positive cells in the appropriate channels ± the standard deviation.
The polynucleotides and polypeptides of the present invention can be used as research reagents and materials for the discovery of treatments and diagnostics for human diseases. This invention provides a method for identification of the receptor for MCP-4. The gene encoding the receptor can be identified by numerous methods known to those skilled in the art, for example, the tracing or panning of the ligand and the FACS classification.
(Coligan, et al., Curren t Protocol s in Immun.
1 (2), Chapter 5, (1991)). Preferably, expression cloning is employed wherein the polyadenylated RNA is prepared from a cell responsive to MCP-4, and a cDNA library created from this RNA is divided into pools and used to transfect COS or other cells. cells that do not respond to MCP-4. Transfected cells that are developed on glass slides are exposed to labeled MCP-4. MCP-4 can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. After fixation and incubation, the slides are subject to
autoradiography analysis. The positive combinations are identified and the sub-combinations are prepared and retransfected using an iterative sub-recombination process and reselection, producing sooner or later a single clone that encodes the putative receptor. As in the alternative method for identification of the receptor, the labeled ligand can be ligated by photoaffinity with the cell membrane or with the extract preparations expressing the receptor molecule. The crosslinked material is resolved by PAGE and exposed to X-ray film. The labeled complex containing the ligand-receptor can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from the microsequencing could be used to design a group of degenerate oligonucleotide probes, to select a cDNA library to identify the gene encoding the putative receptor. This invention also provides a method for selecting compounds, for identifying agonists and antagonists for the polypeptide of the present invention. As an example, a preparation of mammalian cells or a membrane preparation that
expresses an MCP-4 receptor could be contacted with a compound of interest. The ability of the compound to generate a response from a second known messenger system after interaction with the MCP-4 receptor is then measured. Such second messenger systems include, but are not limited to, cAMP-guanylate cyclase, ion channels or phosphoinositide hydrolysis. The ability of a compound to bind to the MCP-4 receptor and elicit a second messenger response identifies that compound as an agonist. A compound that binds but does not elicit a second messenger response identifies that compound as an antagonist. A competitive binding assay may also be employed, in which the compounds are labeled, by radioactivity, to identify the antagonists. Such methods are known in the art. Antagonists include the dominant negative mutants of MCP-4. MCP-4 is a tetrameric polypeptide wherein a mutated unit will cause the entire polypeptide to be non-functional. A dominant negative mutant of MCP-4 binds to the MCP-4 receptor but can not activate cells
(leukocytes and monocytes) to which it binds. An assay to detect the dominant negative mutants of MCP-4 is a chemiotaxis assay in vi tro, wherein a multi-well chemotaxis chamber equipped with polyvinylpyrrolidone-free polycarbonate membranes is used to measure the chemoattractant activity of MCP-4 for leukocytes, in the presence and absence of potential agonist or antagonist molecules. Potential antagonists also include an antibody, or in some cases, an oligopeptide, which binds to the polypeptide and prevents it from binding to its receptor. Another potential antagonist is an antisense construct prepared using antisense technology. Antisense technology can be used to control gene expression through triple helix formation or antisense DNA or RNA, which methods are based on the binding of a polynucleotide to DNA or RNA. For example, the 5 'coding portion of the polynucleotide sequence, which codes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of 10 to 40 base pairs in length. A
DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nuci Aci ds Res. 6: 3073 (1979); Cooney et al., Sci en 241: 456 (1988); ), and Dervan et al, Sci en 251: 1360 (1991)), which prevents the transcription and production of MCP-4. The oligonucleotide of AR? antisense hybridizes to the AR? m in vi, and blocks the translation of the AR? m molecule into the MCP-4 polypeptide (antisense - Okano, J. Neurochem 56: 560 (1991); Oligodeoxinucleotides as Antisense Inhibitors of gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be distributed to the cells, such that the AR? or the AD? Antisense can be expressed in vi to inhibit the production of MCP-4. Potential antagonists include a small molecule that binds to and occupies the active site of the polypeptide, thereby rendering the catalytic site inaccessible to the substrate, such that normal biological activity is prevented. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules.
Antagonists can be used to treat inflammation by preventing the attraction of monocytes to a wound or a trauma site, and to regulate populations of normal lung macrophages, since acute and chronic inflammatory pulmonary diseases are associated with the sequestration of mononuclear phagocytes in the lung. These can also be used to treat rheumatoid arthritis, since it was found that the levels of MCP were significantly elevated in the synovial fluid from patients with rheumatoid arthritis, which suggests that the synovial production of MCP attracts monocytes, whose influence and activation are important in the pathogenesis of degenerative and inflammatory arthropathies. The antagonists can also be used to treat atherosclerosis, since the MCPs are mediators of the infiltration of monocytes in the arterial wall, whose infiltration leads to atherosclerosis, and to prevent allergies, since it has been shown that MCPs directly induce histamine release by basophils. Antagonists can also be used to treat infectious diseases such as tuberculosis, since target or target cells
of tuberculosis, usually monocytes, cause monocytes to release MCPs, which attracts more monocytes to the lungs, causing severe inflammation. Antagonists can be employed in a composition with a pharmaceutically acceptable carrier, for example, as described hereinabove. The polypeptides, and agonists and antagonists, of the present invention may be employed in combination with an appropriate pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide or agonist or antagonist, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes, but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation must be appropriate for the mode of administration. The invention also provides a pharmaceutical package or equipment comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such containers may be a notice in the form prescribed by a government agency, which regulates the manufacture, use or sale of the products.
Pharmaceutical or biological, whose notice reflects the approval by the agency regarding the manufacture, use or sale for human administration. In addition, the polypeptides, or agonists and antagonists of the present invention can be used in conjunction with other therapeutic compounds. The pharmaceutical compositions can be administered in a convenient manner such as by the oral routes, topical, parenteral, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal. The pharmaceutical compositions are administered in an amount that is effective for the treatment and / or prophylaxis of the specific indication. In general, these are administered in an amount of at least about 10 μg / kg of body weight, and in most cases will be administered in an amount not greater than about 8 mg / kg of body weight per day. In most cases, the dose is from about 10 μg / kg to about 1 mg / kg of body weight per day, taking into account the routes of administration, symptoms, etc. Polypeptides and agonists and antagonists that are polypeptides can also be used
according to the present invention, by expressing such polypeptides in vi, which is often referred to as "gene therapy". Thus, for example, cells from a patient can be engineered with a polynucleotide
(DNA or RNA) which encodes an ex vivo polypeptide, with cells engineered which are then provided to a patient to be treated with the polypeptide. Such methods are well known in the art and are apparent from the teachings herein. For example, cells can be engineered by the use of a retroviral plasmid vector containing the RNA encoding a polypeptide of the present invention. Similarly, the cells can be engineered for the expression of an in vivo polypeptide by, for example, procedures known in the art. For example, a packaging cell is transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention, such that the packaging cell now produces infectious viral particles that
they contain the gene of interest. These producer cells can be administered to a patient for genetically engineering the cells in vi and the expression of the polypeptide in vi vo. These and other methods for the administration of a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. Retroviruses from which the retroviral plasmid vectors described herein may be derived include, but are not limited to, the Moloney Murine Leukemia virus, splenic necrosis virus, retroviruses such as Rous Sarcoma Virus. , Harvey's Sarcoma Virus, avian leukosis virus, gibbon monkey leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus. The vector includes one or more promoters. Suitable promoters that can be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller and
collaborators, Bi ot echni q? is 7 (9): 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cell promoters including, but not limited to, histone promoters, pol III, and β-actin). Other viral promoters that can be employed include, but are not limited to, adenoviral promoters, thymidine kinase (TK) promoters, and parvovirus B19 promoters. The selection of an 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 an appropriate promoter. Suitable promoters that can be employed include, but are not limited to, adenoviral promoters, such as the major or major late adenoviral promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the promoter of albumin; the ApoAI promoter; the promoters of human globin; the thymidine kinase promoters
viral, such as the herpes simplex thymidine kinase promoter; Retroviral LTRs (including the modified retroviral LTRs described hereinabove); the β-actin promoter; and the human growth hormone promoters. The promoter can also be the native promoter which controls the gene encoding the polypeptide. The retroviral plasmid vector is used to transduce the packaging cell lines, to form producer cell lines. Examples of packaging cells that can be transfected include, but are not limited to, cell lines PE501, PA317,? -2,? -AM, PAl2, T19-14X, VT-19-17-H2,? CRE, CRIP, GP + E-86, GP + envAml2, and DNA cell lines as described in Miller, Human Gene Therapy, Vol. 1 pages 5-14 (1990), which is incorporated by reference herein. whole. The vector can transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes and calcium phosphate precipitation. In an alternative, the retroviral plasmid vector can be encapsulated within
a liposome, or coupled to a lipid, and then administered to a host. The producer cell line generates infectious particles of the retroviral vector, which include the sequence or nucleic acid sequences encoding the polypeptides. Such retroviral vector particles can then be used to transduce eukaryotic cells, either in vi tro or in vi vo. The transduced eukaryotic cells will express the sequence or nucleic acid sequences encoding the polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic totipotential cells, embryonal carcinoma cells, as well as cells of the hematopoietic line, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells. This invention also relates to the use of the gene of the present invention as a diagnosis. The detection of a mutated form of the gene will allow a diagnosis of a disease or a susceptibility to a disease, which results from the subexpression of MCP-4.
Individuals that possess mutations in the gene of the present invention can be detected at the DNA level by a variety of techniques. The nucleic acids for diagnosis can be obtained from the cells of a patient, including but not limited to blood, urine, saliva, tissue biopsy and autopsy material. Genomic DNA can be used directly for detection, or can be amplified enzymatically by the use of PCR (Saiki et al., Na ture 324: 163-166 (1986)) before analysis. - RNA or cDNA can also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding MCP-4 can be used to identify and analyze the mutations. For example, deletions and insertions can be detected by a change in the size of the amplified product, compared to the normal genotype. Point mutations can be identified by hybridization of the amplified DNA to the radiolabeled RNA or alternatively, to the radiolabelled antisense DNA sequences. The perfectly coupled sequences can be distinguished from the uncoupled duplexes by
digestion with RNAse A, or by differences in the melting temperatures. The sequential differences between the reference gene and the genes that have mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be used as probes to detect specific DNA segments. The sensitivity of this method is greatly increased when combined with PCR. For example, a sequencing primer is used with the double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The determination of the sequence is carried out by conventional procedures with the radiolabelled nucleotide, or by automated sequencing methods, with fluorescent labels. Genetic testing based on DNA sequence differences can be achieved by detecting the alteration of an electrophoretic mobility of DNA fragments on gels with or without denaturing agents. Small deletions and sequential insertions can be visualized by high resolution gel electrophoresis. The different DNA fragments
sequences can be distinguished on denaturing formamide gradient gels, in which the mobilities of different DNA fragments are delayed in the gel at different positions according to their specific melting or partial melting temperatures (see, for example, Myers and collaborators, Sci en 230: 1242 (1985)). Changes -sequential at specific sites can also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (eg, Cotton et al., PNAS USA 85: 4397-4401 (1985)). Thus, the detection of a specific DNA sequence can be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (eg, fragment length polymorphisms). restriction (RFLP)) and Southern blotting or staining of genomic DNA. In addition to more conventional gel electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
The present invention also relates to the diagnostic assay for detecting the altered levels of the polypeptide of the present invention in various tissues, since an overexpression of the proteins compared to normal control tissue samples, can detect the presence of MCP-4. . Assays used to detect the levels of the polypeptide of the present invention in a sample derived from a host are well known to those skilled in the art, and include radioimmunoassays, competitive binding assays, blot analysis or Western blotting, and preferably an ELISA test. An ELISA assay comprises initially the preparation of an antibody specific for the MCP-4 antigen, preferably a monoclonal antibody. In addition, a reporter antibody is prepared against the monoclonal antibody. A detectable reagent such as radioactivity, fluorescence or in this example a horseradish peroxidase enzyme is coupled to the reporter antibody. A sample of a host is now removed and incubated on a solid support, for example, a polystyrene box, which binds the proteins in the sample. Any free protein binding sites on the plate are then
covered by incubation with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the box, during which time the monoclonal antibodies are coupled to any peptide of the present invention coupled to the polystyrene box. Any unbound monoclonal antibody is washed with a buffer. The reporter antibody bound to horseradish peroxidase is now placed in the box, resulting in the binding of the reporter antibody to any monoclonal antibody bound to the polypeptide of the present invention. The uncoupled reporter antibody is then washed. The peroxidase substrates are then added to the box and the amount of color developed in a given period of time is a measurement of the amount of the polypeptide of the present invention, present in a given volume of patient sample, when compared against a standard curve. A competition assay may be employed, wherein the antibodies specific for the polypeptide of the present invention are coupled to a solid support, and labeled MCP-4, and a sample derived from the host is passed over the solid support, and the amount of detected marker, coupled
to the solid support, it can be correlated to an amount of the polypeptide of the present invention in the sample. The sequences of the present invention are also valuable for identification of chromosomes. The sequence is specifically directed towards and can hybridize to a particular site on an individual human chromosome. In addition, there is a current need to identify particular sites on the chromosome. Few chromosome labeling reagents based on effective sequencing data (repeating polymorphisms) are currently available to mark the chromosomal site. The elaboration of the map of the DNAs to the chromosomes according to the present invention is an important first step in the correlation of those sequences with the genes associated with the disease.
Briefly, the sequences can be mapped to the chromosomes by preparing PCR primers (preferably 15-25 base pairs) from the cDNA. Computer analysis of the 3 'untranslated region is used to quickly select the primers
that do not cover more than one exon in the genomic DNA, thus complicating the amplification processes. These primers are then used for the selection by PCR of somatic cell hybrids, which contain individual human chromosomes. Only those hybrids that contain the human gene that corresponds to the primer will produce an amplified fragment. Development of the map by PCR of somatic cell hybrids, is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the oligonucleotide primers, sublocalization can be accomplished with panels of fragments from specific or combined chromosomes of large genomic clones in an analogous manner. Other mapmaking strategies, which can be similarly used to map the chromosome, include in si t u hybridization, preselecting with labeled chromosomes, classified by flow, and pre-selection by hybridization to build chromosome-specific cDNA libraries. In si t u hybridization by fluorescence (FISH) from a cDNA clone to a propagation
chromosomal metaphase, can be used to provide a precise chromosome site in one step. This technique can be used with cDNA as short as 50 or 60 bases. For a review of this technique, see Verma et al., Human Cromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988). Once a sequence has been mapped to an accurate chromosome site, the physical position of the sequence on the chromosome can be correlated with the genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available online through the Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through the analysis of (co-inheritance of physically adjacent genes). Next, it is necessary to determine the difference in the cDNA sequence or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any of
Normal individuals, then the mutation is likely to be the causative agent of the disease. With the current resolution of the elaboration of the physical map and the techniques of the elaboration of the genetic map, a cDNA located precisely for a chromosomal region associated with the disease, could be one of between 50 and 500 potential causal genes. (This assumes a map making resolution of one megabase and one gene per 20 kb). The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them, can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain and humanized antibodies, as well as Fab fragments, or the product of a Fab expression library. Various methods known in the art can be employed for the production of such antibodies and fragments. The antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by means of
direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a non-human. The antibody thus obtained will then bind to the polypeptides themselves. In this way, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies that bind to the complete native polypeptides. Such antibodies can then be used to isolate the polypeptide from the tissue expressing that polypeptide. For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cultures of the cell line can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256: 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72). , and the EBV hybridoma technique to produce human monoclonal antibodies (Colé, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R., Liss, Inc. pp. 77-96). The techniques described for the production of single chain antibodies (American Patent
No. 4,946,778) can be adapted to produce the single chain antibodies for immunogenic polypeptide products of this invention. Also, transgenic mice can be used to express humanized antibodies to the immunogenic polypeptides of this invention. The present invention will be further described with reference to the following examples; however, it should be understood that the present invention is not limited to such examples. All parts or quantities, unless otherwise specified, are by weight. In order to facilitate the understanding of the following examples, certain methods and / or terms of frequent occurrence will be described. "Plasmids" are designated by a p preceded and / or followed by capital letters and / or numbers. The initial plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids according to published procedures. In addition, plasmids equivalent to those described are known in the art and will be apparent to the person of ordinary skill in the art.
"Digestion" of DNA refers to the cleavage or catalytic breakdown of DNA with a restriction enzyme that acts only on certain sequences in DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as it might be known to the person of ordinary skill in the art. For analytical reasons, typically 1 μg of plasmid or DNA fragment with approximately 2 units of enzyme is used in approximately 20 μl of buffer. For the purpose of isolating the DNA fragments for the construction of the plasmid, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. The appropriate buffers and the amounts of substrate for the particular restriction enzymes are specified by the manufacturer. Incubation times of approximately 1 hour at 37 ° C are ordinarily used, but may vary according to the supplier's instructions. After digestion the reaction is subjected to electrophoresis directly on a polyacrylamide gel, to isolate a desired fragment.
The size separation of the excised fragment is performed using 8 percent polyacrylamide gel, described by Goeddel, D. et al., Nucleic Acids Res., 8: 4057 (1980). "Oligonucleotides" refers to either a single chain polydeoxynucleotide or two complementary strands of polydeoxynucleotide, which can be chemically synthesized. Such synthetic oligonucleotides do not have phosphate in the 5 'position and thus will not be ligated to another oligonucleotide without the addition of phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will be ligated to a fragment that has not been dephosphorylated. "Ligature" refers to the process of formation of phosphodiester bonds between two double-stranded nucleic acid fragments (Maniatis, T., et al., Id., Page 146). Unless otherwise provided, ligation can be performed using known buffers and conditions, with 10 units for T4 DNA ligase ("ligase") with only 0.5 μg of approximately equimolar amounts of the DNA fragments. to be linked.
Unless stated otherwise, the transformation was performed as described in the method of Graham, F. and Van der Eb, A. Virology, 52: 456-457 (1973).
Example 1
Bacterial Expression and Purifi cation of MCP-4
the DNA sequence that codes for MCP-4,
ATCC # 75703, is initially amplified using the oligonucleotide PCR primers corresponding to the 5 'and 3' sequence of the processed MCP-4 protein
(minus the sequence of the signal peptide) and the vector sequences in the 3 'direction to the MCP-4 gene. Additional nucleotides corresponding to MCP-4 were added to the 5 'and 3' sequences respectively. The 5 'oligonucleotide primer has the sequence 5'-TCAGGATCCCCTACGGGCTCGTGGTC3' (SEQ ID No. 3), contains a BamH1 restriction enzyme site followed by 18 nucleotides of the MCP-4 coding sequence, starting from the presumed terminal amino acid of the codon of the processed protein. The sequence direction 3 ', 3'-
CGCTCTAGAGTAAAACGACGGCCAGT-5 '(SEQ ID No. 4) contains the sequences complementary to the Xbal site and to a pBluescript SK vector sequence located 3' to the MCP-4 DNA insert. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311)). PQE-9 codes for antibiotic resistance (Ampr), a bacterial origin of replication (ori), a promoter operator regulated by IPTG (P / O), a ribosome binding site (RBS), a 6-His tag and the restriction enzyme sites. PQE-9 was then digested with BamHl and Xbal. The amplified sequences were ligated into pQE-9 and inserted into the structure with the sequence encoding the histidine tag and the RBS. The ligation mixture was then used to transform the E. coli strain ml5 / rep4 available from Qiagen, under the trademark M15 / rep4 by the procedure described in Sambrook, J., et al., Mol ecul ar Cloning: A Labora tory Manual, Cold Spring Laboratory Press, 1989. M15 / rep4 contains multiple copies of plasmid pREP4, which expresses the lacl repressor and also confers resistance to kanamycin (Kanr). The
Transformants are identified by their ability to grow on LB plates and the ampicillin / kanamycin resistant colonies were selected. The plasmid DNA was isolated and confirmed by restriction analysis. Clones containing the desired constructs were grown overnight (O / N) in liquid culture in LB medium supplemented with Amp (100 μg / ml) and Kan (25 μg / ml). O / N culture is used to inoculate a large crop at a ratio of 1: 100 to 1: 250. The cells were developed at an optical density 600 (D.O.b0 °) of between 0.4 and 0.6. IPTG ("isopropyl-B-D-thiogalactopyranoside") was then added to a final concentration of 1 mM. IPTG induces by inactivation of the lacl repressor, the clearance of P / O that leads to the increased expression of the gene. The cells developed an extra 3 to 4 hours. The cells were then harvested by centrifugation. The cell button was solubilized in the chaotropic agent Guanidine 6 Molar hydrochloride. After clarification, the solubilized MCP-4 was purified from this solution by chromatography on a nickel chelate column under conditions that allow strong binding by the proteins containing the 6-His tag. Hochuli, E. and collaborators, J.
Chroma tography 411: 1771-184 (1984). MCP-4 (95% pure) was eluted from the column in 6 molar guanidine hydrochloride, pH 5.0, and for the purpose of renaturation adjusted to 3 molar guanidine hydrochloride, 100 mM sodium phosphate, 10 millimolar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incubation in this solution for 12 hours, the protein was dialyzed to 10 mmolar sodium phosphate.
Example 2
MCP-4 Expression Table in Human Cells
Stain analysis or Northern blotting was carried out to examine the levels of expression of MCP-4 in human cells. The samples of cellular RNA were isolated with the RNAzol ™ system
B (Biotecx Laboratories, Inc. 6023 South Loop East,
Houston, TX 77033). Approximately 10 μg of the total RNA isolated from each specified human tissue were separated on a 1% agarose gel and transferred onto a nylon filter (Sambrook et al., Mol ecul ar Cloning, Cold Spring Harbor
Press, (1989)). The labeling reaction was performed according to the Stratagene Prime-It kit with 50 ng of the
DNA fragment. The labeled DNA was purified with a Select G-50 column (5 Prime-3 Prime, Inc. 5603 Arapahoe Road, boulder, CO 80303). The filter was then hybridized with the full-length MCP-4 gene, radioactively labeled at 1,000,000 cpm / ml in 0.5 M sodium phosphate, pH 7.4 and 7% SDS overnight at 65 ° C. After washing twice at room temperature and twice at 60 ° C with 0.5 x SSC, 0.1% SDS, the filter was then exposed to -70 ° C overnight with an intensification screen. The messenger RNA for MCP-4 is abundant in activated and non-activated T cells, in monocytes and in T cell lines.
Example 3
Cloning and Expression of MCP-4 using the Bacul ovirus Expression System
The DNA sequence encoding the full-length MCP-4 protein, ATCC # 75703, is amplified using the PCR primers corresponding to the 5 'and 3' sequences of the gene:
The amplified sequences were isolated from a 1% agarose gene, using commercially available equipment ("Geneclean", BIO 101 Inc., La Jolla, Ca). The fragment was then digested with restriction endonucleases corresponding to the amplified products, and then purified again on a 1% agarose gel. This fragment is designated F2. The vector pRGl (modification of vector pVL941, discussed below) is used for the expression of the MCP-4 protein using the baculovirus expression system (for review see: Summers, MD and Smith, GE 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555). This expression vector contains the strong polyhedrin promoter of the Autographa cali forni ca nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction endonucleases used to digest the amplified products. The polyadenylation site of simian virus (SV) 40 is used for efficient polyadenylation. For easy selection of the recombinant virus, the E. coli beta-galactosidase gene is inserted into the same
direction than the polyhedrin promoter, followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked on both sides by viral sequences for cell-mediated homologous recombination of the cotransfected, wild-type viral DNA. Many other baculoviral vectors could be used in place of pRG1 such as pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virol. Ogy 170: 31-39). The plasmid is digested with the restriction and dephosphorylated enzymes using calf intestinal phosphatase by methods known in the art. The DNA was then isolated from a 1% agarose gel using commercially available equipment ("Geneclean" BIO 101 Inc., La Jolla, Ca). This vector DNA is designated V2. The F2 fragment and the dephosphorylated plasmid V2 are ligated with the T4 DNA ligase. The HB101 cells of E. coli are then transformed and the bacteria containing the plasmid (pBacMCP-4) are identified with the MCP-4 gene, using the enzymes. The sequence of the cloned fragment is confirmed by DNA sequencing. 5 μg of pBacMCP-4 plasmid are cotransfected with 1.0 μg of a baculovirus
linearized, commercially available ("BaculoGoldMK baculovirus DNA", Farmigen, San Diego, California) using the lipofection method (Felgher et al Proc. Na ti, Acad. Sci. USA 84: 7413-7417 (1987)). of Baculogold ™ viral DNA and 5 μg of plasmid pBacMCP-4 are mixed in a sterile well of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD). 10 μl of Lipofectin plus 90 μl of Grace's medium are mixed and incubated for 15 minutes at room temperature, then the transfection mixture is added dropwise to the Sf9 insect cells (ATCC CRL 1711) seeded on a 35 mm tissue culture with 1 ml of Grace medium without serum.The plate is swung back and forth to mix the newly added solution.The plate is then incubated for 5 hours at 27 ° C. After 5 hours the solution of transfection is withdrawn from The plate is added and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate was placed again in an incubator and the culture was continued for four days at 27 ° c.
After four days the supernatant is collected and a plate assay similar to that described by Summers and Smith (supra) is performed. As a modification, an agar gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used which allows easy isolation of the blue-stained plates. (A detailed description of a "plaque assay" can also be found in the user guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10). Four days after serial dilution, the virus is added to the cells, the plates stained blue are collected with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 μl of Grace medium. The agar is removed by brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect the Sf9 cells seeded in 35 mm boxes. Four days later, the supernatants from these culture boxes are harvested and then stored at 4 ° C. Sf9 cells are grown in Grace's medium supplemented with heat-inactivated FBS,
% The cells are infected with the recombinant baculovirus V-MCP-4 at a multiplicity of infection
(MOI) of 2. Six hours later the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later, 5 μCi of 3bS-methionine and 5 μCi of j5S-cysteine (Amersham) are added. The cells are incubated for 16 hours before they are harvested by centrifugation and the labeled proteins are visualized by SDS-PAGE and autoradiography.
Example 4
Expression Via Gene Therapy
Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue culture medium and separated into small pieces. The small pieces of tissue are placed on a wet surface of a tissue culture flask, approximately 10 pieces are placed in each flask. The flask is turned upside down, closed tightly, and left at room temperature overnight. After 24 hours at room temperature the flask is inverted
and the pieces of tissue remain fixed to the bottom of the flask and fresh medium is added (for example, Ham's F12 medium, with 10% FBS, penicillin and streptomycin). This is then incubated at 37 ° C for about a week. At this time fresh media is added and subsequently it is changed every several days. After an additional period of two weeks in culture, a mono-layer of fibroblasts emerges. The monolayer is trypsinized and is scaled up to higher flasks. pMV-7 (Kirschmeier, PT et al., DNA, 7: 219-25 (1988)) flanked by the long terminal repeats of Moloney murine sarcoma virus, digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase . The linear vector is fractionated on agarose gel and purified, using glass spheres. The cDNA encoding a polypeptide of the present invention is amplified using the PCR primers which correspond to the 5 'and 3' end sequences, respectively. The 5 'primer containing an EcoRI site and the 3' primer further includes a HindIII site. Equal amounts of the linear backbone of murine sarcoma virus are added together
Moloney and the amplified EcoRI and HindIII fragment, in the presence of the T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for the ligation of the two fragments. The ligation mixture is used to transform the HB101 bacteria, which are then plated on agar containing kanamycin, in order to confirm that this vector has the gene of interest properly inserted. The packaging cells pA317 or
Amphotropic GP + aml2 are grown in tissue culture to a confluent density in Dulbecco's modified Eagle medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the medium and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles that contain the gene
(packaging cells are now referred to as producer cells). Fresh media is added to the transduced producer cells, and subsequently, the medium is harvested from a 10 cm plate of the confluent producer cells. The spent medium, which contains the infectious viral particles, is
filtered through a Millipore filter to eliminate the uncoupled producer cells, and this medium is then used to infect the fibroblast cells. The medium is removed from a subconfluent plate of fibroblasts and rapidly replaced with the medium from the producer cells. This medium is removed and replaced with fresh medium. If the virus titer is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector having a selectable marker, such as neo or his. The genetically engineered fibroblasts are then injected into the host, either alone or after being developed to confluence on Cytodex 3 microcarrier spheres. The fibroblasts now produce the protein product.
Example 5
Primal indication of MCP-4 as a Mobili zador de Cél ulas Totipotenci al es de Bone Marrow (Resca te de Bone Marrow)
The effect of MCP-4 on the distribution of primitive hematopoietic progenitors in peripheral blood, spleen and bone marrow was studied in 16-week-old C57B1 / 6 mice (approximately 20 g). In the first experiment, 3 mice were injected intraperitoneally daily with one mg / kg of MCP-4 or saline for 2 days, and analyzed 24 hours after the last injection. In the second experiment, another 3 mice were injected intraperitoneally daily with 1 mg / kg of MCP-4 or saline for 4 days, and analyzed 24 hours after the last injection. In both experiments, the blood of each animal was collected by cardiac puncture and the mice were sacrificed to obtain bone marrow and spleens. The indicated number of cells from each of the tissues was then plated in duplicate in medium containing agar in the presence of 5 ng / ml of IL-3, 50 ng / ml of SCF, 5 ng / ml of M-CSF and 10 ng / ml of IL-la, and incubated for 14 days. In the 2 experiments, the data from the different animals were combined and expressed as the mean ± standard deviation. The results of both experiments show that MCP-4 mobilizes totipotent cells from the bone marrow into the blood
peripheral [Tables 1 and 2]. In the first experiment, after 2 days of treatment with MCP-4, the frequency of HPP-CFC, LPP-CFC and the immature cells in peripheral blood, increased significantly over the controls. No changes were observed in the spleen and there was a significant decrease in
HPP-CFC in the bone marrow [Table 1]. In the second experiment, after • 4 days of treatment with MCP-4, the same significant increase in the frequency of HPP-CFC was observed in the peripheral blood,
LPP-CFC and immature cells. We observed a significant increase in the frequency of immature cells in the spleen, and we observed a significant decrease in PPH-CFC and LPP-CFC in the bone marrow [Table 2]. In particular, it is important to note the presence of immature hematopoietic cells in the peripheral blood after injection of MCP-4.
The effect was observed in the animals treated with MCP-4, which was not due to toxicity, since the FACScan profile of the leukocyte composition of the control mice and the mice treated with MCP-4, is identical [Table 3] .
Example 6
MCP-4 as a Me the oppressor Against the Chitosan Arabinoside
In this experiment, Lin cells (1 x 10f 'cell / ml) were plated in a growth medium that was supplemented with 5 ng / ml mouse IL-3, 50 ng / ml mouse SCF (column 1 ); IL-3, SCF and 100 ng / ml of MCP-4 (column 2); or IL-3, SCF and
100 ng / ml irrelevant protein HG200-3-B
(column 3). After 48 hours of incubation, a group of the above cultures received 50 μg / ml of
Ara-C and incubation was continued for an additional 24 hours. The cells were then harvested, washed three times with HBSS to remove the drug and the cytokines, and assayed for the presence of HPP-CFC and LPP-CFC as described in the legend of the Figure. The results are expressed as the% protection average (± standard deviation). The% protection was calculated as follows: the percentage protection is expressed as the number of colonies found in the cultures incubated in the presence of Ara-C, divided by the number of colonies found in the cultures incubated without Ara-C x 100.
Data from one of 3 experiments, as shown in Table 6. All samples were tested in duplicate.
Example 7
MCP-4 as a Myeloprotector against 5-fl uorouracil or
A mononuclear population of mouse bone marrow cells was extracted from cells confined to a line, by negative selection using a panel of monoclonal antibodies directed against cell surface antigens. The resulting population of cells (Lin cells) was resuspended (1 x 10; cell / ml) in a growth medium containing IL-3 (5 ng / ml), SCF (50 ng / ml), GM-CSF (5 ng / ml), M-CSF (5 ng / ml) and IL -la (10 ng / ml) and 1 ml of this cell suspension was dispersed in culture tubes. 1) A group of cultures in duplicate did not receive chemokine; 2) cultures in duplicate with MCP-4 at 100 ng / ml; and 3) cultures in duplicate with an irrelevant protein at 100 ng / ml. All cultures were incubated in a tissue culture incubator for 48 hours, at which point one culture of each group received 5-
fluorouracil at 100 μg / ml, and incubation was continued for an additional 24 hours. All cultures were then harvested, washed three times with HBSS, and then tested for the presence of HPP-CFC and LPP-CFC as described in the legend of Figure 5. Percentage protection is expressed as the number of colonies detected in the cultures incubated in the presence of 5-FU, divided by the number of colonies found in the incubated cultures without 5-FU x 100. The data are expressed as the mean ± the standard deviation. Two experiments were performed and each test was in duplicate. See Figure 7.
Example 8
Effect of MCP-4 on the Neuronal Survival Corti cal
Sprague-Dawley rats on day 17 of gestation were sacrificed, and the cortex was removed and the meninges were carefully excised from the pieces of cortical tissue. Simple cell suspensions were prepared and the cells were plated out in medium containing 5% horse serum at a density of
,000 cells / well. After 24 hours the medium containing serum was removed and the serum free medium was added to the cultures. Included in the serum-free cultures had a concentration of MCP-4 as shown in Figure 8. The MCP-4 used is an MCP-4 polypeptide encoded by the polynucleotide sequence as shown in SEQ ID No. 1 of the request. The medium was changed every day and MCP-4 was added again. Neurons were maintained in culture for 6 days before the viability test. Cell viability was evaluated using the live / dead test kit of Molecular Probes. This assay is a two-color fluorescence cell viability assay, based on the simultaneous determination of living cells and dead cells. The living cells are distinguished by the presence of the ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the calcein AM that permeates the almost non-fluorescent cells, to intensely fluorescent calcein. Polycationic calcein is retained very well by living cells, and thus produces an intense uniform green fluorescence in living cells. In this way, the emission reading
(approximately 530 nm) is a measure of the total number of cells in the cultures. As shown in Figure 8, the number of living cells increased as the concentration of MCP-4 increased. Numerous modifications and variations of the present invention are possible in the light of the foregoing teachings and, therefore, within the scope of the appended claims, the invention may be practiced in a manner other than that which is particularly described.
LIST OF SEQUENCES
GENERAL INFORMATION i) APPLICANT: A) NAME: Human Genome Sciences, Inc. B) STREET: 9410 Key West Avenue C) CITY: Rockville D) STATE: Maryland E) COUNTRY: United States of America F) POSTAL CODE (ZIP): 20850-3338 G) TELEPHONE: 301-309-8504 H) TELEFAX: 301-309-8512 ii) TITLE OF THE INVENTION: Protein 4 Monocyte Chemoattractant iii) SEQUENCE NUMBER: 6 iv) COMPUTER LEGIBLE FORM: A) TYPE MEDIUM: Flexible Disk B) COMPUTER: PC compatible with IBM C) OPERATING SYSTEM: PC-DOS / MS-DOS D) SOFTWARE: Patentin Relay # 1.0, Version # 1.30 (EPO) v) DATA OF THE CURRENT APPLICATION: A) APPLICATION NUMBER: (to be assigned) B) DATE OF SUBMISSION: JUNE 07, 1996
vi) DATA FROM THE PREVIOUS APPLICATION: A) NUMBER OF APPLICATION: US 08 / 479,126 B) DATE OF SUBMISSION: JUNE 07, 1995
2) INFORMATION FOR SEQ ID No. 1: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 360 base pairs B) TYPE: nucleic acid C) TYPE OF HEBRA: simple D) TOPOLOGY: linear ii) TYPE OF MOLECULE: DNA (genomic) ix) CHARACTERISTICS: A) NAME / KEY: CDS B) LOCATION: 1.357
xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 1:
ATG GCA GGC CTG ATG ACC ATA GTA ACC AGC CTT CTG TTC CTT GGT GCT 48 Met Wing Gly Leu Met Thr He Val Thr Ser Leu Leu Phe Leu Gly Val 1 5 10 15
TGT GCC CAC CAC ATC ATC CCT ACG GGC TCT GTG GTC ATA CCC TCT CCC 96 Cys Ala His His He He Pro Pro Thr Gly Ser Val Val He Pro Ser Pro 20 38 30
TGC TGC ATG TTC TTT GTT TCC AAG AGA ATT CCT GAG AAC CGA GTG GTC 144 Cys Cys Met Phe Phe Val Ser Lys Arg He Pro Glu Asn Arg Val Val 35 40 45
AGC TAC CAG CTG TCC AGC AGG AGC AC TGC CTC AAG GGA GGA GTG ATC 192 Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Gly Gly Val Lie 50 55 60
TTC ACC ACC AAG AAG GGC CAG CAG TTC TGT GGC GAC CCC AAG CAG GAG 240 Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro Lys Gln Glu 65 70 75 80
TGG GTC CAG AGG TAC ATG AAG AAC CTG GAC GCC AAG CAG AAG AAG GCT 298 Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln Lys Lys Ala 85 90 95
TCC CCT AGA GCC AGG GCA GTG GCT GTC AAG GGC CCT GTC CAG AGA TAT 336 Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly Pro Val Gln Arg Tyr 100 105 110
CCT GGC AAC CA ACC ACC TGC TAA 360 Pro Gly Asn Gln Thr Thr Cys 115
2) INFORMATION FOR SEQ ID No. 2: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 119 amino acids B) TYPE: amino acid D) TOPOLOGY: linear ii) TYPE OF MOLECULE: protein xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 2:
Met Wing Gly Leu Met Thr Lie Val Thr Ser Leu Leu Phe Leu Gly Val
1 5 10 15
Cys Ala Hls His Lie He Pro Thr Gly Ser Val Val Lie Pro Ser Pro 20 25 30
Cys Cys Met Phe Phe Val Ser Lys Arg He Pro Glu Asn Arg Val Val 35 40 45
Being Tyr Gln Leu Being Being Arg Being Thr Cys Leu Lys Gly Gly Val Lie 50 55 60
Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro Lys Gln Glu 65 70 75 80
Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Wing Lys Gln Lys Lys Wing
Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly Pro Val Gln Arg Tyr 100 105 110
Pro Gly Asn Gln Thr Thr Cys 115
2) INFORMATION FOR SEQ ID No. 3: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 28 base pairs B) TYPE: nucleic acid C) TYPE OF HEBRA: simple D) TOPOLOGY: linear ii) TYPE OF MOLECULE: DNA (genomic) xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 3 TCAGGATCCC CTACGGGCTC GTGTGGTC 28
2) INFORMATION FOR SEQ ID No. 4: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 26 base pairs B) TYPE: nucleic acid C) TYPE OF HEBRA: simple D) TOPOLOGY: linear
ii) TYPE OF MOLECULE: DNA (genomic) xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 4: TGACCGGCAG CAAAATGAGA TCTCGC 26 5 2) INFORMATION FOR SEQ ID No. 5: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 99 amino acids B) TYPE: amino acid 0 C) TYPE OF HEBRA: simple D) TOPOLOGY: linear ii) TYPE OF MOLECULE: protein xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 5:
Met Lys Val Ser Ala Ala Leu Leu Cys Leu Leu Leu He Ala Ala Thr 1 5 10 15
Phe He Pro Gln Gly Leu Wing Gln Pro Asp Wing He Asn Wing Pro Val 20 25 30
Thr Cys Cys Tyr Asn Phe Thr Asn Arg Lys He Ser Val Gln Arg Leu 35 40 45
Wing Ser Tyr Arg Arg He Thr Ser Ser Lys Cys Pro Lys Glu Wing Val
50 55 60
He Phe Lys Thr He Val Wing Lys Glu He Cys Wing Asp Pro Lys Gln 65 70 75 80
Lys Trp Val Gln Asp Ser Met Asp His Leu Asp Lys Gln Thr Gln Thr 85 90 95
Pro Lys Thr
2) INFORMATION FOR SEQ ID No. 6: i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 93 amino acids B) TYPE: amino acid C) TYPE OF HEBRA: simple D) TOPOLOGY: linear ii) TYPE OF MOLECULE: protein xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 6:
Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala 1 5 10 15
Leu Cys Asn Gln Val Leu Be Ala Pro Leu Ala Wing Asp Thr Pro Thr 20 25 30
Wing Cys Cys Pro Ser Tyr Thr Ser Arg Gln He Pro Gln Asn Phe He 35 40 45
Wing Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val He 50 55 60
Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Wing Asp Pro Ser Glu Glu 65 70 75 90
Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Wing 85 90
It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.
Having described the invention as above, property is claimed as contained in the following:
Claims (20)
1. An isolated polynucleotide, characterized in that it comprises a member selected from the group consisting of: a) a polynucleotide encoding the polypeptide comprising amino acid 26 to amino acid 93 as described in SEQ ID No. 2; b) a polynucleotide encoding the polypeptide comprising amino acid 1 to amino acid 93 as described in SEQ ID No. 2; c) a polynucleotide encoding a mature polypeptide having the amino acid sequence expressed by the DNA contained in ATCC Deposit No. 75703; d) a polynucleotide capable of hybridizing to, and which is at least 70% identical to, the polypeptide of (a), (b) or (c); and e) a polynucleotide fragment of the polynucleotide of (a), (b), (c) or (d).
2. The polynucleotide according to claim 1, characterized in that the polynucleotide is DNA.
3. The polynucleotide according to claim 2, characterized in that it encodes the polypeptide comprising amino acid 1 to 93 of SEQ ID No. 2.
4. A vector, characterized in that it contains the DNA according to claim 2.
5. A host cell, characterized in that it is engineered with the vector according to claim 4.
6. A process for the production of a polypeptide, characterized in that it comprises: the expression from the host cell according to claim 5, of the polypeptide encoded by said DNA.
7. A process for the production of cells capable of expressing a polypeptide, characterized the process because the cells are genetically manipulated with the vector according to claim 4.
8. A polypeptide, characterized in that it comprises a member selected from the group consists of i) a polypeptide having the deduced amino acid sequence of SEQ ID No. 2 and fragments, analogs and derivatives thereof; and ii) a polypeptide encoded by the ATCC cDNA Deposit No. 75703 and fragments, analogs and derivatives of said polypeptide.
9. The polypeptide according to claim 8, characterized in that the polypeptide comprises amino acid 1 to amino acid 93 of SEQ ID No. 2.
10. A compound, characterized in that it inhibits the activation of the polypeptide according to claim 8.
11. A method for the treatment of a patient in need of MCP-4, characterized in that the method comprises: administering to the patient a therapeutically effective amount of the polypeptide according to claim 8.
12. The method according to claim 11, characterized in that the therapeutically effective amount of the polypeptide is administered by provision to the patient, of the DNA encoding the polypeptide, and expressing the polypeptide in vi vo.
13. A method for the treatment of a patient in need of inhibiting an MCP-4 polypeptide, characterized in that the method comprises: administering to the patient a therapeutically effective amount of the compound according to claim 10.
14. A process for diagnosing a disease or a susceptibility to a disease related to an under-expression of MCP-4, characterized the process because it comprises: determining a mutation in a nucleic acid sequence encoding MCP-4.
15. A diagnostic process, characterized in that it comprises: the analysis of the presence of the polypeptide according to claim 8, in a sample derived from a host.
16. A method for identifying compounds that bind and inhibit the activation of the polypeptide according to claim 8, characterized in that the method comprises: contacting a cell expressing on the surface thereof, a receptor for the polypeptide, the receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor, with a compound under conditions that allow binding to the receptor; and detecting the absence of a signal generated from the interaction of the compound with the receptor.
17. A method for identifying compounds that bind to, and inactivate the polypeptide according to claim 8, characterized in that the method comprises: contacting a cell expressing on the surface thereof, a receptor for the polypeptide, the receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor, with a compound under conditions that allow binding to the receptor; and detecting the absence of a signal generated from the interaction of the compound with the receptor.
18. A method for increasing the amount of hematopoietic progenitor cells in the peripheral blood of a host, characterized in that the method comprises: administering to the host the polypeptide according to claim 8, in an amount effective to increase the amount of hematopoietic progenitor cells in the peripheral blood of the host.
19. A method for inhibiting the destruction of hematopoietic progenitor cells, resulting from the treatment of a host with a chemotherapeutic agent, characterized in that it comprises: administering to the host the polypeptide according to claim 8, in an amount effective to inhibit the destruction of cells hematopoietic progenitors by a chemotherapeutic agent.
20. A method for inhibiting the degeneration of neuronal cells in a host, characterized in that the method comprises: administering to a host in need thereof the polypeptide according to claim 8, in an amount effective to inhibit the degeneration of neuronal cells .
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/479,126 US5866373A (en) | 1995-04-21 | 1995-06-07 | Polynucleotide encoding a human chemotactic protein |
US08479126 | 1995-06-07 | ||
PCT/US1996/010087 WO1996040762A1 (en) | 1995-06-07 | 1996-06-07 | Monocyte chemotactic protein-4 |
Publications (2)
Publication Number | Publication Date |
---|---|
MX9709192A MX9709192A (en) | 1998-03-31 |
MXPA97009192A true MXPA97009192A (en) | 1998-10-15 |
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