Tandem cDNAs encoding chemokines CC-1, CC-2, and CC-3

Details Tandem cDNAs encoding chemokines CC-1, CC-2, and CC-3 Tandem cDNAs encoding chemokines CC-1, CC-2, and CC-3

1998-12-30

2001-01-30

Fitzgerald, David L. (Department: 1646)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C530S350000, C530S351000, C514S002600, C424S085100

Reexamination Certificate

active

06180773

ABSTRACT:

The present invention relates to the nucleic acid of a tandem gene coding for two polypeptides of the cytokine class, to cytokines CC-2 and CC-3 and their biologically active fragments and/or derivatives, to a medicament containing the peptides according to the invention, to a diagnostic agent, to the use of cytokines CC-2 and CC-3 for medicinal indications, and to a nucleic acid probe which will hybridize to a polynucleotide coding for cytokines CC-2 or CC-3 or a fragment thereof.
The CC type chemokines belong to a family of polypeptides which have proven to be mediators of immune reactions, and they have recently attracted attention due to their antiviral activity with respect to HIV.
The chemokines are small, basic, heparin-binding polypeptides which participate in the induction of immune reactions and in inflammatory processes (1, 2). The first members of this family were cloned from stimulated tonsillary lymphocytes or macrophages in the middle of the eighties. The family is subdivided into CXC and CC chemokines according to the relative positions of the first two cysteine residues. Most of the CXC and CC chemokines exhibit chemotaxis with respect to cells which participate in immune reactions. Some CC chemokines show multiple biological activities, such as control of the proliferation and differentiation of hematopoetic progenitor cells (5, 6, 7, 8, 9), activation and attraction of a wide range of immune cells (10, 11), and postulated participation in HIV-1 and HIV-2 replication control in CD4+ T lymphocytes (12). The most frequently described and best studied CC chemokines in the above described context include MIP-1&agr;, MIP-1&bgr; and RANTES. The subdivision of the chemokines into CC and CXC chemokines is also reflected in the structure and position of their genes on the chromosomes (14, 15, 16, 17). Genes of human CXC chemokines are generally found on chromosome 4 and consist of four exons and three introns, whereas the genes of CC chemokines lie on the human chromosome 17 and exhibit a conserved structure with three exons and two introns (18, 19). In 1995, a new type of chemokines has been discovered: the so-called C family which is represented by murine lymphotactin (20). This finding shows that the chemokines are not restricted to only two subfamilies.
The present invention relates, inter alia, to the cloning of a human tandem gene, and to the corresponding mature bicistronic mRNA which contains the open reading frames (ORFs) for a new CC chemokine with six cysteine residues, CC-2, and for the above described factor HCC-1. This factor was isolated from human hemofiltrate, and it has been shown to circulate in high levels in the human plasma of healthy subjects (1-5 nM) and of patients suffering from renal failure (5-80 nM) (21). Unlike other, related CC chemokines, HCC-1 fails to exhibit chemoactivity towards monocytes, and it only stimulates slightly the intracellular Ca
2+
mobilization, and the release of enzymes in these cells. Further, it was reported that HCC-1 promoted the proliferation of CD34+ bone marrow stem cells in a dose-dependent way (21). Despite of the activities described for HCC-1, its major biological function remains unclear.
The nucleic acid of a human tandem gene according to the invention codes for two new CC type chemokines the sequences of which are somewhat homologous with that of MIP-1&agr;.
Thus the present invention relates to nucleic acids corresponding to the tandem gene having the nucleotide sequences shown in SEQ ID NO: 1, encoding the chemokines designated CC-2 (SEQ ID NO: 6) and CC-1 (SEQ ID NO: 7), and in SEQ ID NO: 2, encoding chemokines CC-2 (SEQ ID NO: 11) and CC-3 (SEQ ID NO: 12).
The transcription of the tandem gene leads to a bicistronic mature transcript which contains the non-overlapping open reading frames for the recently described factor HCC-1 and for CC-2. Moreover, alternative splicing of the primary transcript yields at least one additional CC type chemokine, cytokine CC-3. The present invention also relates to these new CC type cytokines as well as their biologically active amidated, acetylated, phosphorylated and/or glycosylated derivatives.
Two functional promoter regions were identified within the tandem gene. Their ability to induce the expression of the tandem gene was examined. The acquired data provide some basic knowledge about the structure and expression of the new human CC-2/HCC-1 tandem gene and describe a mechanism according to which the coexpression of closely linked genes could be regulated in higher eucaryotes.
The present invention also relates to a medicament containing cytokine CC-2 and/or CC-3 as well as their biologically active amidated, acetylated, phosphorylated and/or glycosylated derivatives as the active ingredient. The medicament is administered in accordance with appropriate galenics, adapted to its way of administration. As usual with peptides, the medicament according to the invention can be administered via parenteral, intravenous, intramuscular, intranasal or bucal routes. The amount of peptide to be administered is between 10 and 3000 &mgr;g per unit dose.
The diagnostic agent according to the invention contains polyclonal or monoclonal antibodies against the peptide according to the invention, optionally in a fluorescence-labeled or radiolabeled form, to be employed in per se known assays such as ELISA or RIA.
The diagnostic agent according to the invention may contain nucleic acids coding for the cytokines, mRNA, the nucleic acid according to the invention or fragments thereof, or poly- or oligonucleotides capable of hybridizing to the nucleic acid coding for the cytokines CC-2 and/or CC-3.
The peptides cytokine CC-2 and/or CC-3 according to the invention can be used for the preparation of a medicament for the treatment of disorders of cell migration, diseases of the immune system, tumors and dysfunction of regulatory growth functions.
The present invention also relates to nucleic acid probes which are capable of hybridizing to the nucleic acid according to the invention under stringent conditions.
The full length cDNA of the sequence of the bicistronic CC-2/HCC-1 cDNA was obtained by 5′-RACE PCR using 5 &mgr;g of whole RNA from T-84 cells. The PCR fragment was directly sequenced and reamplified from the first strand of adult liver cDNA using an oligo-dT primer in combination with a specific primer (35 cycles); this was followed by a second amplification step (30 cycles) with nested primers. A computer-aided sequence analysis confirmed the presence of two open reading frames (ORFs) within the cDNA. The upstream ORF codes for a hypothetical precursor with a length of 113 amino acids (aa). Interestingly, the translation of the first ORF is terminated by three stop codons. A hydrophobicity plot yielded an N-terminal hydrophobic sequence of 20 aa which could be a leader peptide. The sequence of the hypothetical peptide which we called CC-2 is 46% homologous with that of MIP-1&agr;, but contains two additional cysteine residues. The downstream ORF codes for HCC-1. Alternative splicing within intron 1 of the primary transcript yields a peptide variation of HCC-1 which contains 16 additional amino acids.
The structure of the human CC-2/HCC-1 tandem gene has been determined. The 20 kbp tandem gene which was isolated from a &lgr;-FIX-II phage which had been isolated from a human DNA library contains seven exons, of which exons &agr;-&dgr; code for the hypothetical peptide CC-2, and exons 1-3 code for HCC-1. The whole region was mapped by restriction analysis. The order and orientation of the SstI restriction fragments was determined by sequencing the flanking regions of each SstI restriction site. The sequencing was performed using an automated fluorescence sequencer. Restriction fragments containing the HCC-1 gene were detected by Southern blotting with the HCC-1 cDNA as a radioactive probe. The fifteen terminal bp of the monocistronic HCC-1 cDNA (box hatched in black upstream from exon 1) do not appear in the bicistronic mRNA; therefore, they are at the same tim

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