Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1997-07-29
2002-12-17
McKelvey, Terry (Department: 1636)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C536S023400, C536S023720, C536S024100
Reexamination Certificate
active
06495526
ABSTRACT:
BACKGROUND OF THE INVENTION
The cell division cycle is one of the most fundamental processes in biology which, in multicellular organisms, ensures the controlled generation of cells with specialized functions. Under normal growth conditions, cell proliferation is tightly regulated in response to diverse intra- and extracellular signals. This is achieved by a complex network of proto-oncogenes and tumor-suppressor genes that are components of various signal transduction pathways. Activation of a proto-oncogene(s) and/or a loss of a tumor suppressor gene(s) can lead to the unregulated activity of the cell cycle machinery. This, in turn, will lead to unregulated cell proliferation and to the accumulation of genetic errors which ultimately will result in the development of cancer (Pardee, Science 246:603-608, 1989).
In the eukaryotic cell cycle a key role is played by the cyclin-dependent kinases (CDKs). Cdk complexes are formed via the association of a regulatory cyclin subunit and a catalytic kinase subunit. In mammalian cells, the combination of the kinase subunits (such as cdc2, CDK2, CDK4 or CDK6) with a variety of cyclin subunits (such as cyclin A, B1, B2, D1, D2, D3 or E) results in the assembly of functionally distinct kinase complexes. The coordinated activation of these complexes drives the cells through the cell cycle and ensures the fidelity of the process (Draetta, Trends Biochem. Sci. 15:378-382, 1990; Sherr, Cell 73:1059-1065, 1993). Each step in the cell cycle is regulated by a distinct and specific cyclin-dependent kinase. For example, complexes of Cdk4 and D-type cyclins govern the early G1 phase of the cell cycle, while the activity of the CDK2/cyclin E complex is rate limiting for the G1 to S-phase transition. The CDK2/cyclin A kinase is required for the progression through S-phase and the cdc2/cyclin B complex controls the entry into M-phase (Sherr, Cell 73:1059-1065, 1993).
The CDK complex activity is regulated by mechanisms such as stimulatory or inhibitory phosphorylations as well as the synthesis and degradation of the kinase and cyclin subunit themselves. Recently, a link has been established between the regulation of the activity of cyclin-dependent kinases and cancer by the discovery of a group of CDK inhibitors including the p27
Kip1
, p21
Waf1/Cip1
and p16
Ink4/MTS1
proteins. The activity of p21
Waf1/Cip1
is regulated transcriptionally by DNA damage through the induction of p53, senescence and quiescence (Harper et al., Cell 75:805-816, 1993). The inhibitory activity of p27
Kip1
is induced by the negative growth factor TGF-&bgr; and by contact inhibition (Polyak et al., Cell 78:66-69, 1994). These proteins, when bound to CDK complexes, inhibit their kinase activity, thereby inhibiting progression through the cell cycle. Although their precise mechanism of action is unknown, it is thought that binding of these inhibitors to the CDK/cyclin complex prevents its activation. Alternatively, these inhibitors may interfere with the interaction of the enzyme with its substrates or its cofactors.
While p21
Waf1/Cip1
and p27
Kip1
inhibit all the CDK/cyclin complexes tested, p16
Ink4/MTS1
, p15, p18 and p19 block exclusively the activity of the CDK4/cyclin D and CDK6/cyclin D complexes in the early G1 phase (Serrano et al., Nature 366:704-707, 1993), by either preventing the interaction of Cdk4 and Cyclin D1, or indirectly preventing catalysis. As mentioned above, the p21
Waf1/Cip1
is positively regulated by the tumor suppressor p53 which is mutated in approx. 50% of all human cancers. p21
Waf1/Cip1
may mediate the tumor suppressor activity of p53 at the level of cyclin-dependent kinase activity. p16
Ink4/MTS1
is the product of a tumor suppressor gene localized to the 9p21 locus, which is frequently mutated in human cancer cells.
Of all the various kinases, the CDK4/cyclin D complexes are known to play an important role in regulating cell cycle progression in early G1. These complexes function as integrators of various growth factor-induced extracellular signals and as a link between the different signal transduction pathways and other cyclin-dependent kinases. The expression of the cyclin D1 positive regulatory subunit, is deregulated by gene translocations, retroviral insertions and amplifications in parathyroid adenomas, lymphomas, esophageal and breast carcinomas. The targeted overexpression of cyclin D1 in the mammary epithelium of transgenic mice induces mammary adenomas and adenocarcinomas. This confirms that cyclin D1, when overexpressed, acts as an oncogene (Wang et al., Nature 369:669-671, 1994). These data supports the idea that the lack of functional p16
Ink4/MTS1
or the overexpression of cyclin D1 leads to the deregulation of CDK4/cyclin D1 kinase activity and thereby contribute to uncontrolled cell proliferation.
The prominent role of CDK/cyclin kinase complexes, in particular, CDK4/cyclin D kinase complexes, in the induction of cell proliferation and their deregulation in tumors, makes them ideal targets for developing highly specific anti-proliferative agents.
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to a nucleic acid comprising a nucleotide sequence encoding a chimeric polypeptide having at least two CDK-binding motifs derived from different proteins which bind to cyclin dependent kinases (CDKs). The chimeric polypeptide binds to CDKs and inhibits cell-cycle progression.
The chimeric polypeptide can be a fusion protein, or can be generated by chemically cross-linking the CDK-binding motifs.
In preferred embodiments, at least one of the CDK-binding motifs is a CDK-binding motif of a CDK inhibitor protein, such as an INK4 protein, e.g., p15, p16, p18 and p19, or a CIP protein, e.g., p21
CIP1
, p27
KIP1
, and p57
KIP2
. However, it will be understood that other CDK-binding motifs may be useful. Indeed, the CDK-binding motif of the INK4 proteins is characteristized by tandemly arranged ankyrin-like sequences, which sequences exist in other proteins and, for those which are able to bind a CDK, can be used to generate the subject chimeric proteins. Likewise, the CDK-binding motif can be a p21/p27 inhibitory domain of a protein which has some homology with the CIP protein family. Exemplary chimeric proteins of the present invention are designated by SEQ ID No:2, 5 and 7, and are encoded by the CDS's designated in SEQ ID No:1, 4 and 6.
In preferred embodiments, the CDK-binding motifs of the chimeric protein have different binding specificities, relative to one and other, for cyclin dependent kinases. For instance, the chimeric protein can be generated with a CDK-binding motif from a protein which binds to and inhibits a CDK involved in progression of the cell cycle in G
0
and/or G
1
phase, and another CDK-binding motif from a protein which binds to and inhibits a CDK involved in progression of the cell cycle in S, G
2
and/or M phase. That is, the chimeric protein will bind to and inhibit a plurality (two or more) of cyclin dependent kinases which are active in different phases of the cell-cycle.
In most embodiments, the nucleic acid will further include a transcriptional regulatory sequence for controlling transcription of the nucleotide sequence encoding the chimeric polypeptide, e.g., the transcriptional regulatory sequence is operably linked to a chimeric gene encoding the chimeric polypeptide. For example, the present invention specifically contemplates recombinant transfection systems which include: (i) a gene construct including a nucleic acid encoding a chimeric polypeptide comprising CDK-binding motifs from two or more different proteins which bind to cyclin dependent kinases, and operably linked to a transcriptional regulatory sequence for causing expression of the chimeric polypeptide in eukaryotic cells, and (ii) a gene delivery composition for delivering the gene construct to a cell and causing the cell to be transfected with the gene construct. For example, the gene construct can be derived from a viral vector, such as an adenoviral vector, an adeno-associated viral vector or a retroviral vecto
Beach David H.
Gyuris Jeno
Lamphere Lou
GPC Biotech Inc.
McKelvey Terry
Ropes & Gray
Sandals William
LandOfFree
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