Use of protein H as cytostatic agent

Details Use of protein H as cytostatic agent Use of protein H as cytostatic agent

2000-05-11

2004-12-21

Canella, Karen A. (Department: 1642)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S300000, C530S350000, C530S825000

Reexamination Certificate

active

06833355

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the use of bacterial proteins. It is based on the finding that protein H, which can be derived from
Streptococcus pyogenes,
has a cytostatic effect on eukaryotic cells, specifically murine B-lymphocytes. The present invention therefore relates to the use of protein X, and fragments or derivatives thereof, as cytostatic agents, especially in the treatment of diseases involving undesired cell proliferation.
BACKGROUND TO THE INVENTION
Protein H can be obtained from
Streptococcus pyogenes,
as described in EP-A-0 371, 199 and WO 91/19740. These publications also provide the amino acid sequence of protein H from
Streptococcus pyogenes
and the sequence of the DNA encoding it. Protein H has a characteristic spectrum of immunoglobulin-binding properties, as described in EP-A-0 371,199 and it is also capable of binding albumin (WO 91/19740). In WO 91/19740, a number of regions within protein H were identified, and designated S, A, B, C1, C2, C3 and D regions. Albumin-binding activity was found to be located in the C and/or D regions.
SUMMARY OF THE INVENTION
We have now found that protein H has a further, and unexpected, property. When biotinylated protein H was incubated with murine B-lymphocytes, it was found that protein H was targeted to the cell nucleus, and inhibited cell proliferation in a dose-dependent manner, although without inducing apoptosis (cell death). Thus, protein H has a cytostatic effect on these cells. We have also observed nuclear targeting of protein H in human T-lymphocytes (Jurkat cells). This suggests that the cytostatic effect may not be confined to B-lymphocytes, but may extend to other eukaryotic cell types as well.
In the cell, protein H was found to interact with actin, and with nucleophosmin/B23, a protein known to shuttle between the nucleus and cytoplasm. In the nucleus itself, protein H was found to interact additionally with the nuclear proteins SET and hnRNP A2/B1, resulting in nuclear accumulation of protein H. We believe that protein H penetrates the cell membrane, becomes associated with nucleophosmin/B23 in the cytoplasm and is transported across the nuclear membrane into the nucleus, where it interacts with SET and hnRNP. As protein H interacts with actin, the actin cytoskeleton may be involved in transporting protein H from the inside of the cell membrane into the cytoplasm, where it becomes associated with NPM/B23. However, the possibility that protein H simply diffuses through the cytoplasm cannot be excluded.
We believe that the interaction with NPM/B23 and/or SET and/or hnRNP A2/B1 may be responsible for the cytostatic effect, although the precise mechanism is not yet fully elucidated. Interactions between protein H and other nuclear components, e.g. nuclear proteins, may also be involved.
The finding that protein H has cytostatic properties was surprising in view of the previously known properties of protein H. Previously, protein H has been known as an immunoglobulin-binding protein, located at the surface of the Streptococcus bacterium, where it protects the bacterium by blocking complement activation at the bacterial cell surface by means of its interaction with the Fc region of IgG. It is therefore surprising that it also appears to effect a further virulence function on behalf of the Streptococcus bacterium, namely a cytostatic effect. It is also surprising that protein H is apparently capable of exerting this effect from the cell nucleus when it has previously been known to exert unrelated effects at the cell surface. By contrast, protein A, an immunoglobulin-binding bacterial cell surface molecule derived from
Staphlyococcus aureus,
showed no nuclear accumulation. Proteins A and H are functionally similar in that both bind to the same site in the Fc region of IgG. The fact that the functionally similar protein H showed nuclear accumulation is therefore all the more surprising.
Based on our findings, protein H, and fragments and derivatives thereof, can be used to combat undesired cell proliferation, and therefore to treat diseases where undesired cell proliferation takes place. Treatment of tumours of various kinds is one preferred possibility.
Accordingly, the invention provides:
Use of protein H, or a fragment or derivative thereof which is capable of exerting a cytostatic effect on a eukaryotic cell, in the manufacture of a medicament for exerting a cytostatic effect on a eukaryotic cell.
The invention also provides:
A method of exerting a cytostatic effect on a eukaryotic cell comprising administering to said cell an effective non-toxic amount of protein H or a fragment or derivative thereof which is capable of exerting a cytostatic effect on said eukaryotic cell.


REFERENCES:
patent: 371 199 (1990-06-01), None
patent: WO 91/19740 (1991-12-01), None
Burgess et al “Possible Dissociation of the Heparin-binding and Mitogenic Acitivies of Heparin-binding Growth Factor-1 from Its Receptor-binding Activities by Site-directed Mutagenesis of a single Lysine Residue”, Journal of Cellular Biology, 1990, vol. 1.*
Lazar et al, “Transforming Growth Factor Alpha: Mutation of Aspartic Acid 47 and Leuine 48 Results in Different Biological Activities”, Molecular and Cellular Biology, 1988, vol. 8, pp. 1247-1252.*
Genbank Accession No:NP_047206, haemagglutinin protein H.*
Ciaiolo et al, “Enhancement of methotrexate cytotoxicity by modulation of proliferative activity in normal and neoplastic T lymphocytes and in a myeloid leukemia cell line”, Tumori, 1988, vol. 74, pp. 537-542.*
Hultsch et al, “The effect of immunophilin ligands rapamycin and FK506 on the proliferation of mast cells and other hemaopoietic cell lines”, Molecular Biology of the Cell, 1992, vol. 3, pp. 981-987.*
Balkwill, FR, “Tumor necrosis factor”, British Medical Bulletin, 1989, vol. 45, pp. 389-400.*
Reiger et al, Glossary of Genetics and Cytogenetics, 4th edition, 1976, pp. 17-18.*
Mathews and Van Holde, Biochemistry (textbook), 2nd edition, 1996, pp. 165-171.*
Matthews, B., “Genetic and structural analysis of the protein stability problem”, in Perspectives in Biochemistry, Hans Neurath, Ed., 1989, vol. 1, pp. 6-9.*
Axcrona et al., “Multiple Ligand Interactions for Bacterial Immunoglobulin-Binding Proteins on Human and Murine Cells of the Hematopoetic Lineage,”Scandinavian Journal of Immunology 42(3):359-367, 1995.
Frick et al., “Protein H—a surface protein ofStreptococcus pyogeneswith separate binding sites for IgG and albumin,”Molecular Microbiology 12(1): 143-151, 1994.
Identification and Characterization of SET, a Nuclear Phosphoprotein Encoded by the Translocation Break Point in Acute Undifferentiated Leukemia: Authors(s) Yoshifumi Adachi et al., The Journal of Biological Chemistry, (Jan. 1994), vol. 269, No. 3 pages, 2256-2262.
HnRNP Proteins and the Biogenesis of mRNA: Authors (s) Gideon Dreyfuss et al., Annu. Rev. Biochem (1990); vol. 62, pp. 289-321.
Major Nucleolar Proteins Shuttle between Nucleus and Cytoplasm: Authors(s) R.A. Borer et al, Cell, (Feb. 10, 1989) vol. 56, pp. 379-390.

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