Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-01-17
2004-10-26
Ungar, Susan (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023500, C435S320100, C435S325000, C435S366000, C435S252300
Reexamination Certificate
active
06809189
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to the field of apoptosis. Apoptosis is an active and programmed physiological process for eliminating superfluous, altered or malignant cells (Earnshaw, 1995; Duke et al., 1996). Apoptosis is characterized by shrinkage of cells, segmentation of the nucleus, condensation and cleavage of DNA into domain-sized fragments in most cells followed by internucleosomal degradation. The apoptotic cells fragment into membrane-enclosed apoptotic bodies. Finally, neighboring cells and/or macrophages will rapidly phagocytose these dying cells (Wylie et al., 1980; White, 1996). Cells grown under tissue-culture conditions and cells from tissue material can be analyzed for being apoptotic with agents staining DNA, for example, DAPI, which stains normal DNA strongly and regularly, whereas apoptotic DNA is stained weakly and/or irregularly (Noteborn et al., 1994; Telford et al., 1992).
The apoptotic process can be initiated by a variety of regulatory stimuli (Wyllie, 1995; White, 1996; Levine, 1997). Changes in the cell survival rate play an important role in human pathogenesis of diseases, for example, in cancer development and autoimmune diseases, where enhanced proliferation or decreased cell death (Kerr et al., 1994; Paulovich, 1997) is observed. A variety of chemotherapeutic compounds and radiation have been demonstrated to induce apoptosis in tumor cells, in many instances via wild-type p53 protein (Thompson, 1995; Bellamy et al., 1995;Steller, 1995; McDonell et al., 1995).
Many tumors, however, acquire a mutation in p53 during their development, often correlating with poor response to cancer therapy. Certain transforming genes of tumorigenic DNA viruses can inactivate p53 by directly binding to it (Teodoro, 1997). An example of such an agent is the large T antigen of the tumor DNA virus SV40. For several (leukemic) tumors, a high expression level of the proto-oncogene Bcl-2 or Bcr-abl is associated with a strong resistance to various apoptosis-inducing chemotherapeutic agents (Hockenberry 1994; Sachs and Lotem, 1997).
For such tumors lacking functional p53 (representing more than half of the tumors), alternative antitumor therapies are under development based on induction of apoptosis independent of p53 (Thompson 1995; Paulovich et al., 1997). One has to search for the factors involved in induction of apoptosis, which do not need p53 and/or can not be blocked by antiapoptotic activities, such as Bcl-2 or Bcr-abl-like ones. These factors might be part of a distinct apoptosis pathway or might be (far) downstream of the apoptosis inhibiting compounds.
Apoptin is a small protein derived from chicken anemia virus (CAV; Noteborn and De Boer, 1995; Noteborn et al., 1991; Noteborn et al., 1994, 1998a) which can induce apoptosis in human malignant and transformed cell lines, but not in untransformed human cell cultures. In vitro, Apoptin fails to induce programmed cell death in normal lymphoid, dermal, epidermal, endothelial and smooth-muscle cells. However, when normal cells are transformed they become susceptible to apoptosis by Apoptin. Long-term expression of Apoptin in normal human fibroblasts revealed that Apoptin has no toxic or transforming activity in these cells (Danen-van Oorschot, 1997 and Noteborn, 1996).
In normal cells, Apoptin was found predominantly in the cytoplasm, whereas in transformed or malignant cells, i.e., characterized by hyperplasia, metaplasia or dysplasia, it was located in the nucleus, suggesting that the localization of Apoptin is related to its activity (Danen-van Oorschot et al. 1997).
Apoptin-induced apoptosis occurs in the absence of functional p53 (Zhuang et al., 1995a) and cannot be blocked by Bcl-2, Bcr-abl (Zhuang et al., 1995), or the Bcl-2-associating protein BAG-1 (Danen-Van Oorscho, 1997a; Noteborn, 1996).
Therefore, Apoptin is a therapeutic compound for the selective destruction of tumor cells or other hyperplasia, metaplasia or dysplasia, especially for those tumor cells which have become resistant to (chemo)-therapeutic induction of apoptosis due to the lack of functional p53 and (over)-expression of Bcl-2 and other apoptosis-inhibiting agents (Noteborn and Pietersen, 1998). It appears, that even premalignant, minimally transformed cells are sensitive to the death-inducing effect of Apoptin In addition, Noteborn and Zhang (1998) have shown that Apoptin-induced apoptosis can be used to diagnose cancer-prone cells and to treat cancer-prone cells.
The fact that Apoptin does not induce apoptosis in normal human cells, at least not in vitro, shows that a toxic effect of Apoptin treatment in vivo will be very low. Noteborn and Pietersen (1998) and Pietersen et al. (1999) have provided evidence that adenovirus expressed Apoptin does not have an acute toxic effect in vivo. In addition, in nude nice it was shown that Apoptin has a strong antitumor activity.
However, to further enlarge the array of therapeutic anticancer or antiautoimmune disease compounds available in the art, additional therapeutic compounds are desired that are designed to work alone, sequentially to, or jointly with Apoptin, especially in those cases wherein p53 is (partly) nonfunctional.
DISCLOSURE OF THE INVENTION
The invention provides novel therapeutic possibilities, for example, novel combinatorial therapies or novel therapeutic compounds that can work alone, sequentially to, or jointly with Apoptin, especially in those cases wherein p53 is (partly) nonfunctional.
In a first embodiment, the invention provides an isolated or recombinant nucleic acid or functional equivalent or fragment thereof encoding an Apoptin-associating proteinaceous substance capable of providing apoptosis, alone or in combination with other apoptosis inducing substances, such as Apoptin. Proteinaceous substance is herein defined as a substance comprising a peptide, polypeptide or protein, optionally having been modified by, for example, glycosylation, myristilation, phosphorylation, the addition of lipids, by homologous or heterologous di- or multimerisation, or any other (posttranslational) modifications known in the art.
Apoptin-associating is herein defined as belonging to the cascade of substances specifically involved in the cascade of events found in the apoptosis pathway as inducable by Apoptin, preferably those substances that are specifically involved in the p53-independent apoptosis pathway.
In a preferred embodiment, the invention provides an isolated or recombinant nucleic acid or functional equivalent or fragment thereof encoding an Apoptin-associating proteinaceous substance capable of providing apoptosis derived from a cDNA library, preferably a vertebrate cDNA library, such as derivable from poultry, but more preferably a mammalian cDNA library, preferably wherein said cDNA library comprises human cDNA. An Apoptin-associating proteinaceous substance obtained by determining a vertebrate analogue (preferably human) of an Apoptin-associating proteinaceous substance derived from an invertebrate cDNA library is also included.
In another embodiment, the invention provides an isolated or recombinant nucleic acid or functional equivalent or fragment thereof encoding an Apoptin-associating proteinaceous substance capable of providing apoptosis capable of hybridizing to a nucleic acid molecule encoding an Apoptin-associating proteinaceous substance capable of providing apoptosis as shown in
FIG. 1
,
2
,
5
and/or
7
A-
7
D, in particular encoding a novel protein capable of providing apoptosis or functional equivalent or functional fragment thereof called Apoptin-associating protein 2 or 3, abbreviated herein also as AAP-2 or AAP-3.
FIGS. 1 and 2
show an approximately 1100 and 900 bp fragment of the AAP-2 fragment as depicted in
FIGS. 7A-7D
. All 3 nucleotide sequences encode a protein with at least the capability of binding to Apoptin and providing apoptosis. Of course, an isolated or recombinant nucleic acid or functional equivalent or fragment thereof encoding an additional Apoptin-associating proteinaceous substance capable of associating with the
Danen-van Oorschot Astrid Adriana A. M.
Noteborn Mathieu Hubertus M.
Rohn Jennifer Leigh
Weiss Bertram
Leadd B.V.
TraskBritt
Ungar Susan
LandOfFree
Apoptin-associating protein does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Apoptin-associating protein, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Apoptin-associating protein will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3326778