Stem cell inhibiting proteins

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...

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435 695, 4353201, 435325, 4352523, 530350, 530351, 536 235, C12N 121, C07H 2102, C07K 1452

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060571233

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BRIEF SUMMARY
This invention relates to proteinaceous compounds having the properties of inhibitors of stem cell proliferation. In particular, the invention relates to engineered variants of protein molecules with stem cell inhibition activity, their preparation and pharmaceutical compositions containing them and their use as adjuncts to chemotherapy or radiotherapy, for example in the treatment of cancer.
The diverse cells of the haemopoietic system are derived from multipotential stem cells by a process of sequential division and differentiation. The proliferation of the stem cell population is controlled in part by an inhibitory molecule produced by bone marrow macrophages (Lord et al., Brit. J. Haematol. 34 441, (1976)). The murine haemopoietic stem cell inhibitor has been shown to be an 8 kDa protein, macrophage inflammatory protein-1 alpha (MIP-1.alpha. (Graham et al., Nature 344 442, (1990)). The properties of the stem cell inhibitor include protecting stem cells from the toxic effects of cell cycle specific cytotoxic agents (Lord and Wright, Blood Cells, 6 581 (1980)). Stem cell inhibitors therefore have enormous clinical potential as agents to protect the stem cells from the chemotherapy or radiotherapy regimes used in tumour therapy. Additionally, stem cell inhibitors may be used in the treatment of hyperproliferative diseases such as psoriasis, either alone or in conjunction with cytotoxic agents. Amino acid sequence homologies suggested that either the human LD78 or ACT2 gene products were the human homologues of the murine stem cell inhibitor (FIG. 1a) (Schall, Cytokine 3 165-183 (1991)). It has been demonstrated that the human LD78 gene product is the functional homologue of murine MIP-1.alpha., (Pragnell CRC Beatson Laboratory Scientific Report pp 21-25, (1990), Dunlop et al., Blood 79: 2221-2225 (1992)).
As a component of the present invention, the secondary and tertiary structure of LD78 and MIP-1.alpha. have been shown to be almost identical. Only a difference in the nature of a side-chain or charge interaction in the vicinity of Trp-57 is observed for the two proteins. Despite having a similar secondary structure to LD78 and MIP-1.alpha., near u.v. c.d. studies show ACT2 has a different tertiary conformation as highlighted by the shape and intensity of the spectrum. This provides strong evidence that LD78 and not ACT2 is the human bomologue of MIP-1.alpha..
A major problem shared by murine MIP-1.alpha. and human LD78, which limits their potential clinical utility, is that at concentrations as low as 25 .mu.g/ml in physiological ionic strength buffer they form large soluble multimeric complexes which have a tendency to aggregate. The native MIP-1.alpha. and LD78 protein molecules have a molecular weight of 7,966 Da and 7,712 Da respectively. For both proteins, the soluble multimeric complexes show a broad heterogeneous mixture of molecular weights ranging from 100,000 Da to >>200,000 Da. The principal consequence of the multimerisation and aggregation phenomena is that clinical administration of the protein is compromised. Aggregation and multimerisation can lead to varying efficacy, impaired tissue penetration and enhanced immunogenicity. Another important shortcoming is that, during production and formulation, aggregation will result in heterogeneous pharmaceutical preparations.
Cloudy aggregates are often observed upon reconstitution of pure, lyophilised LD78 or MIP-1.alpha. protein in physiological ionic strength buffer at pH 7.4. Aggregates are removed by centrifugation prior to further analysis. Size exclusion chromatography (Comparative Example 3) of the soluble reconstituted MIP-1.alpha. and LD78 following clarification show that the majority of the protein chromatographs as broad peaks of molecular weights of 100,000-800,000 Da. The broad trailing edge of the peaks demonstrates the existence of a range of high molecular weight complexes for each protein. The size exclusion profile of MIP-1.alpha. also reveals a population of tetrameric molecules in equilibrium with the large multimers.
A

REFERENCES:
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Graham et al., Nature, 344:422 (1990).
Schall, Cytokine, 3:165-183 (1991).
Dunlop et al., Blood, 79:2221-2225 (1992).
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W. R. Paukovits, et al., "Hemoregulatory Peptide pGlu-Glu-Asp-Cys-Lys: A New Synthetic Derivative for Avoiding Dimerization and Loss of Inhibitory Activity," Molecular Pharmacology, 38:401-409 (Jun. 1990).

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