Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-05-23
2000-12-12
Mertz, Prema
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 711, 435 712, 4353201, 435325, 435471, 4352523, 43525411, 530351, 536 231, 536 235, 514 2, 514 8, 514 12, 514826, 514885, 514886, C12N 510, C12N 1519, C07K 1452, A61K 3819
Patent
active
061597112
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to derivatives of RANTES and their uses.
The protein known as RANTES was originally cloned by Schall T. J. et al., (J. Immunol. 141 1018-1025 (1988)) in Krensky's laboratory at Stanford University School of Medicine. The term RANTES is derived from the phrase "Raised on activation, normal T-cell derived and secreted" (relevant letters underlined). Its expression is inducible by antigen stimulation or mutagen activation of T-cells. The protein is a member of the chemokine superfamily (Schall T. J., Cytokine 3 165-183 (1991); Oppenheim, J. J. et al., Ann. Rev. Immunol. 9 617-48 (1991)). The pure protein was first identified in 1992 in platelets (Kameyoshi et al., J. Exp. Med. 176 587-592 (1992)). It is a potent attractor for eosinophils, CD4.sup.+ CD45RO.sup.+ T-cells, and also for monocytes. It has a sixty-eight amino acid sequence.
A receptor for RANTES has recently been cloned (Gao, J. L. et al., J. Exp. Med. 177 1421-7 (1993); Neote, K., et al., Cell 72 415-25 (1993))--and this has been shown to bind chemokines in the rank order of potency of MIP-1.alpha.>RANTES.
The present invention provides polypeptides which are antagonists of RANTES and/or of MIP-1.alpha..
Despite the considerable interest in cytokines generally and the work discussed above on RANTES and RANTES receptors in particular, prior to the present invention there has been no disclosure of the above antagonists or of the possible utilities of such antagonists.
According to the present invention there is provided a polypeptide having substantial amino acid sequence homology with RANTES and functioning as an antagonist to RANTES and/or MIP-1.alpha. in respect of one or more of the following: to MIP-1.alpha.; RANTES and/or due to the presence of MIP-1.alpha.; and cells.
The polypeptides provided by the present invention are useful in further characterising RANTES and its effects--for example in studying RANTES induced chemotaxis, mobilisation of calcium ions and receptor binding. They are also useful in the characterisation of the binding of RANTES to its receptors. They are useful in studying MIP-1.alpha. for corresponding reasons.
Additionally, the polypeptides of the present invention are believed to be useful in the treatment of various diseases, as will be discussed later.
A preferred polypeptide of the present invention acts as an antagonist to RANTES and/or to MIP-1.alpha. due to the presence of one or more N-terminal amino acids (which are not present at the corresponding position in RANTES and which can therefore be regarded as additional N-terminal amino acids relative to those present at the N-terminus of RANTES). These N-terminal amino acids are preferably naturally occurring (L-) amino acids (which can be incorporated by using recombinant DNA techniques or by peptide fusion techniques). However non-naturally occurring amino acids (e.g. D-amino acids) may be used. These may be incorporated by using chemical synthesis techniques.
There may be only one such additional amino acid, in which case it may be Leucine or Methionine, for example. Such polypeptides can be prepared by any suitable techniques (e.g. by using gene cloning techniques, chemical synthesis, etc.). In one embodiment of the present invention they are prepared by providing a larger polypeptide comprising a desired sequence and then using enzymatic cleavage to produce a polypeptide consisting of the desired sequence.
The polypeptides of the present invention may comprise more than one additional N-terminal amino acids e.g. they may include up to five, up to ten or up to twenty additional amino acids. In some cases over twenty additional N-terminal amino acids may be present.
Again, any suitable techniques can be used to prepare such polypeptides.
The various aspects of the present invention will now be discussed in further detail below.
The present inventors have discovered that using an E. coli expression system intended to express RANTES in a form corresponding to mature human RANTES (i.e. with the signal sequence removed) a polypeptide
REFERENCES:
Cunningham et al. (1989) Science vol. 266, pp. 1081-1085.
George et al (1988) Macromolecular Sequencing & Synthesis. Selected Methods & Applications chap. 12, pp. 127-149. Alan R. Liss, Inc. N.Y.
Zhang et al, "Structure/Activity Analysis of Human Monocyte Chemoattractant Protein-1 (MCP-1) by Mutagenesis", Journal of Biological Chemistry 269(22):15918-15924 (1994).
Gong and Clark-Lewis, "Antagonists of Monocyte Chemoattractant Protein-1 Identified by Modification of Functionally Critical NH-2 terminal Residues", Journal of Experimental Medicine 181:631-640 (1995).
Herbert et al, "Scanning Mutagenesis of Interleukin-8 Identifies a Cluster of Residues Required for Receptor Binding", 266(28):18989-18994 (1991).
Moser et al, "Interleukin-8 Antagonists Generated by N-terminal Modification", Journal of Biological Chemistry 268(10):7125-7128 (1993).
Proudfoot Amanda E. I.
Wells Timothy N. C.
Glaxo Group Limited
Mertz Prema
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