Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Patent
1996-01-29
1999-08-10
Draper, Garnette D.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
424 851, 536 235, 435 695, 435 691, 530350, 530395, C07K 1452, C12N 1519
Patent
active
059360674
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to variants of stem cell inhibitors.
The treatment of cancer with chemotherapeutic agents is designed to attack and destroy cells which are undergoing division within the body. A side effect of such treatment is thus the destruction of normal cells, particularly the stem cells of the haematopoietic system and the epithelial stem cells which line the scalp and gut. Radiation can also cause similar destruction of such cells.
It has been proposed that in order to improve the treatment of cancers by chemotherapy it would be desirable to protect stem cells from cell cycle specific cytotoxic drugs. WO89/10133 discloses a stem cell inhibitor and describes the use of the inhibitor in the treatment of cancers. The inhibitor may be administered to a patient in order to protect stem cells during chemotherapy.
Stem Cell Inhibitor (SCI), also known as MIP1-.alpha. is a peptide of about 8 kD which forms large self aggregates, the molecular weight of which is dependent upon the concentration of SCI/MIP1-.alpha. monomers (Graham et al, 1990, Nature 344;442, Wolpe & Cerami, 1989, FASEB J, 3; 2656). It has been found that SCI/MIP1-.alpha. has a native, aggregated molecular weight of about 100 kD at 0.1 mg/ml in physiological buffers such as PBS. It has been found that diluting SCI/MIP1-.alpha. to about 20-100 ng/ml or less will bring about disaggregation of this protein.
Human SCI/MIP1-.alpha. has been cloned by us (Graham et al (1992), Growth Factors 7;151-160). The cDNA has also been cloned by Nakao et al (1990, Mol. Cell, Biol., 10;3646-58) and called LD78.beta.. A variant of the cDNA LD78.alpha. was also found, which has a very similar sequence. It differs by only 4 amino acid residues. The human cDNA and protein sequence of the factor cloned by us is shown is Seq. ID No. 1. The first 27 amino acids are a leader sequence. The mature protein starts at residue 28 (ala). The amino acid sequence of the variant found by Nakao et al is shown as Seq. ID No. 3. The leader sequence of the protein is one amino acid shorter and thus the mature protein starts at residue 27 (ala). The sequence of the murine homologue, upon which we have conducted our work, is also known and is very similar. It can be found for example in Graham et al (1994, J. Biol. Chem., 269; 4974-78).
It has been reported (Mantel et al, 1993, PNAS 90;2232) that monomeric SCI/MIP1-.alpha. is more active than the aggregated form in inhibiting in vitro and in vivo stem cell proliferation. In using SCI/MIP1-.alpha. in the treatment of humans it would be desirable to administer monomeric protein, not just from an activity point of view but also in order to provide reliable and reproducible formulations. However, it is likely that the low concentrations of SCI/MIP1-.alpha. which must be made in order to provide monomeric protein will be too low for use in practice.
We have now surprisingly found that it is possible to obtain SCI/MIP1-.alpha. variants which retain substantially the activity of the native protein but which do not form the same large aggregates. These mutants are stable as monomers or as small conglomerates (eg dimers or tetramers) at concentrations many fold higher than native SCI/MIP1-.alpha.. Thus for those variants which have activity comparable to native SCI/MIP1-.alpha., the variants may have higher activity in vivo on a unit weight basis.
Accordingly, the present invention provides a Stem Cell Inhibitor protein which comprises at least one amino acid alteration from its native form which does not significantly aggregate but which retains substantially unaltered stem cell inhibitory activity. The protein may comprise either the full length stem cell inhibitor or the mature processed form lacking the leader sequence.
The invention also provides pharmaceutical compositions comprising a stem cell inhibitor according to the invention in combination with a pharmaceutically acceptable carrier or diluent, and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the
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Graham Gerard
Pragnell Ian
Brown Scott A.
DesRosier Thomas J.
Draper Garnette D.
Genetics Institute Inc.
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