Drug – bio-affecting and body treating compositions – Lymphokine
Reexamination Certificate
2000-08-17
2003-04-29
Mertz, Prema (Department: 1646)
Drug, bio-affecting and body treating compositions
Lymphokine
C514S002600, C514S008100, C514S012200, C514S824000, C514S825000, C514S826000, C514S885000, C514S886000
Reexamination Certificate
active
06555105
ABSTRACT:
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 Krenskyp'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
+
CD45RO
+
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&agr;>RANTES.
The present invention provides polypeptides which are antagonists of RANTES and/or of MIP-1&agr;.
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&agr; in respect of one or more of the following:
(a) the chemotaxis of THP-1 cells in response to RANTES and/or in response to MIP-1&agr;;
(b) the mobilisation of calcium ions in THP-1 cells due to the presence of RANTES and/or due to the presence of MIP-1&agr;; and
(c) the binding of RANTES and/or of MIP-1&agr; to receptors of THP-1 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&agr; 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&agr; 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 was expressed in which an additional N-terminal methionine was present (this was not cleaved from the remaining sequence by endogenous
E. coli
proteases). It was surprisingly found that the presence of this additional amino acid substantially changed the characteristics of the polypeptide relative to those of RANTES. The methionylated polypeptide (referred to herein as methionylated RANTES or Met-RANTES) was found to act as an antagonist of RANTES and of MIP-1&agr; in various assays and was not found to have any substantial agonist activity.
It should be appreciated that in many cases where N-terminal methionylation occurs in
E. coli,
it makes little or no difference to a polypeptide's properties or results in it merely having reduced levels of its previous biological activity. It was therefore totally unexpected that N-terminal methionylation would in the case of the present invention actually result in antagonistic activity.
In order to determine whether or not this effect was limited to the present of an N-terminal methionine, another polypeptide was produced, in which the N-terminal methionine was replaced with an N-terminal Leucine. Again, this was found to act as an antagonist of RANTES, as was a further polypeptide in which the N-terminal methionine was replaced with an N-terminal Glutamine.
In a preferred form, the polypeptide of the present invention has the sequence:
(i) MSPYSSDT TPCCFAYIAR PLPRAHIKEY FYTSGKCSNP AVVFVTRKNR QVCANPEKKW VREYINSLEM S (sometimes referred to herein as “Met-RANTES”)
(ii) LSPYSSDT TPCCFAYIAR PLPRAHIKEY FYTSGKCSNP AVVFVTRKNR QVCANPEKKW VREYINSLEM S (sometimes referred to herein as “Leu-RANTES”)
or
(iii) QSPYSSDT TPCCFAYIAR PLPRAHIKEY FYTSGKCSNP AVVFVTRKNR QVCANPEKKW VREYINSLEM S (sometimes referred to herein as “Gln-RANTES”)
or has a sequence which is substantially homologous with any of the above sequences. The polypeptide may be in a glycosylated or unglycosylated form.
The term “substantially homologous” when used herein includes amino acid sequences having at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence homology with the given sequence (in order of preference). This term can include, but is not limited to, amino acid sequences having from 1 to 20, from 1 to 10 or from 1 to 5 single amino acid deletions, insertions or substitutions relative to a given sequence—provided that the resultant polypeptide acts as an antagonist to RANTES or to MIP-1&agr;.
The polypeptide may be in substantially pure form. It may be isolated from naturally occurring polypeptides.
It should be noted that it is well known in the art that certain amino acids can be replaced with others resulting in no substantial change in the properties of a polypeptide. Such possibilities are within the scope of the present invention.
It should also be noted that deletions or insertions of amino acids can often be made which do not substantially change the properties of a polypeptide. The present invention includes such deletions or insertions (which may be, for example up to 10, 20 or 50% of the length of the specific antagonists sequence given above). The present invention also includes within its scope fusion proteins in which the polypeptides of the present invention are fused to another moiety. This may be done, for example, for the purpose of labelling or for a medicinal purpose.
The present inventors have demonstrated that a polypeptide of the present invention can act as an antagonist to the effects of RANTES or of MIP-1&agr; in chemotaxis, calcium mobilisation and receptor binding in THP-1 cells (a monocytic cell line). These cells are available from ATCC (American Tissue Culture Collection) and act as a good model system for studying RANTES because they show calcium responses and chemotactic responses to RANTES and MIP-1&agr;, a
Proudfoot Amanda E. I.
Wells Timothy N. C.
Glaxo Group Limited
Mertz Prema
Nixon & Vanderhye P.C.
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