Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-04-06
2004-03-30
Tate, Christopher R. (Department: 1654)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C530S300000, C530S350000, C514S008100
Reexamination Certificate
active
06713280
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of therapeutics and diagnostics, and in particular to the delivery of biological molecules and other chemical substances into the interior of cells.
BACKGROUND OF THE INVENTION
Many biological molecules and pharmaceutical agents must first traverse the cell membrane in order to exert an action on cells. Transmembrane delivery of nucleic acids, for example, has relied on protein carriers, antibody carriers, liposomal delivery systems, direct injection into cells, electroporation, cell fusion, viral delivery, and calcium-phosphate mediated transformation.
Another method of transmembrane delivery of exogenous molecules, including nucleic acids, has been receptor-mediated endocytosis. This involves conjugating the biological or pharmaceutical agent with a ligand which specifically binds to receptors on a cell membrane. The process of endocytosis is initiated or activated by the binding of the ligand to the receptor. Receptor-mediated endocytosis has been utilized for delivery of proteins as well as nucleic acids to cells. Generally, the ligand is chemically conjugated by covalent, ionic or hydrogen bonding to the exogenous molecule of interest that is still recognized in the conjugate by the target receptor. Conjugation of exogenous molecules of interest to ligand substances having corresponding cell surface receptors is described in U.S. Pat. No. 5,108,921. In particular, the method of the '921 patent relies upon the transmembrane transport of exogenous materials across a membrane having biotin or folate receptors that initiate transmembrane transport of receptor bound species.
International patent application PCT/US90/01002 (WO 90/104448) of Genentech, Inc., discloses covalent conjugates of oligonucleotides and lipids for securing transmembrane delivery of the oligonucleotide into cells. Examples of such lipids include fatty acids and esters thereof, glycerides, e.g., triglycerides, glyceryl ethers, phospholipids, sphingolipids, fatty alcohols, waxes, terpenes, and steroids. The lipids may be naturally derived or synthetically prepared.
International patent application PCT/US90/05272 (WO 91/04753) of Cetus Corporation, describes conjugates of antisense oligonucleotides and ligand-binding molecules which recognize a cell surface receptor. The ligand-binding molecule is a growth factor, an antibody to a growth factor, or an antibody to a cell surface receptor.
U.S. Pat. No. 5,550,111 discloses conjugates of 2′, 5′-oligoadenylate and an adduct which results in enhanced penetration into cells. The adduct may comprise a vitamin selected from those vitamins which have a corresponding cell receptor on targeted mammalian cells. Such vitamins include for example, vitamin B12, biotin, riboflavin or folic acid. Alternatively, the adduct may comprise a lipophilic molecule or radical, such as an acyl group of the formula —OC(CH
2
)
x
CH
3
, wherein x is an integer from 1 to 20, preferably from 2 to 14.
Frequently, useful effectors of intracellular targets comprise proteinaceous substances such as peptides and polypeptides. For example, the product of the Bcl-2 gene is known to contribute to neoplastic cell expansion by preventing normal cell turnover caused by physiological cell death mechanisms. The Bcl-2 gene product is an intracellular protein. Bcl-2 (B cell lymphomalleukemia 2) was originally identified at the chromosomal breakpoint of t(14;18)-bearing B-cell lymphomas. Bcl-2 is now known to belong to a growing family of proteins which regulate programmed cell death or apoptosis. The Bcl-2 family includes both death antagonists (Bcl-2, Bcl-x
L
, Bcl-w, Bfl-1, Brag-l, Mcl-l and Al) and death agonists (Bax, Bak, Bcl-x
5
, Bad, Bid, Bik and Hrk) (Thompson,
Science
267:1456-62 (1992); Reed,
J. Cell Biol
. 124:1-6 (1994); Yang et al.,
Blood
88:386401 (1996)). This family of molecules shares four homologous regions termed Bcl homology (BH) domains BH1, BH2, BH3, and BH4. All death antagonist members contain the BH4 domain while the agonist members lack BH4. It is known that the BH1 and BH2 domains of the death antagonists such as Bcl-2 are required for these proteins to heterodimerize with death agonists, such as Bax, and to repress cell death. On the other hand, the BH3 domain of death agonists is required for these proteins to heterodimerize with Bcl-2 and to promote apoptosis.
Programmed cell death or apoptosis plays a fundamental role in the development and maintenance of cellular homeostasis. Homologous proteins and pathways in apoptosis are found in a wide range of species, indicating that cellular demise is critical for the life and death cycle of the cell in all organisms. When extracellular stimuli switch on the cell-death signal, the response of the cell to such stimuli is specific for the particular cell type (Bonini et al., Cell 72:379-95 (1993)). The pathway to cellular suicide is controlled by certain checkpoints (Oltvai,
Cell
79:189-92 (1994)). The Bcl family proteins, including both antagonists of apoptosis (such as Bcl-2) and agonists of apoptosis (such as Bax), constitute the primary checkpoint. As such, the transmission of a cell-death signal can be either promoted or blocked by the different combinations of the Bcl-2 family members. The three-dimensional structure of a death antagonist, Bcl-X
L
, as determined by X-ray crystallography and NMR spectroscopy, provides a structural basis for understanding the biological functions of Bcl-2 family members and for developing novel therapeutics targeting Bcl-2 mediated apoptotic pathways (Muchmore et al.,
Nature
381:335-41 (1996)).
The detailed mechanism of Bcl-2 proteins in mediating molecular pathways of apoptosis has been the subject of intensive investigation. It is known that the apoptotic signaling pathway involves the activation of caspases which, once activated, cleave several cellular substrates such as poly(adenosine diphosphate-ribose) polymerase (PARP) and lead to final events of apoptosis. Bcl-2 plays a crucial role in regulating the process of apoptosis. One possible mechanism for Bcl-2 function is that Bcl-2 inhibits the release of cytochrome c from mitochondria. Cytochrome c is important for the activation of caspases. As such, Bcl-2 blocks caspase activation and subsequent events leading to apoptosis.
Being able to block apoptosis, Bcl-2 is known to contribute to neoplastic cell expansion by preventing normal cell turnover caused by physiological cell death mechanisms. High levels and aberrant patterns of Bcl-2 gene expression are found in a wide variety of human cancers, including ~30-60% of prostate, ~90% of colorectal, ~60% of gastric, ~20% of non-small cell lung cancers, ~30% of neuroblastomas, and variable percentages of melanomas, renal cell, and thyroid cancers, as well as acute and chronic lymphocytic and non-lymphocytic leukemias (Ellis et al.,
Cell Biol
. 7, 663 (1991); Henkart,
Immunity
1, 343 (1994)); Kägi et al.,
Science
265, 528 (1994); Kägi et al.,
Nature
369, 31 (1994); Heusel et al.,
Cell
76, 977 (1994)).
The expression levels of Bcl-2 protein also correlate with relative resistance to a wide spectrum of current chemotherapeutic drugs and ?-irradiation (Hanada et al.,
Cancer Res
. 53:4978-86 (1993); Kitada et al.,
Antisense Res. Dev
. 4:71-9 (1994); Miyashita et al.,
Cancer Res
. 52:5407-11 (1992); Miyashita et al.,
Blood
81:151-7 (1993)). Since Bcl-2 can protect against such a wide variety of drugs which have very different mechanisms of action, it is possible that all these drugs use a common final pathway for the eventual induction of cell death which is regulated by Bcl-2. This notion is supported by the findings that chemotherapeutic drugs induce cell death through a mechanism consistent with apoptosis as opposed to necrosis. Therefore, Bcl-2 can inhibit the cell killing effect of currently available anticancer drugs by blocking the apoptotic pathway.
Because of its role in blocking apoptosis, Bcl-2 plays an important role in many types of cancer. As noted above, Bcl-2 blocks apoptosis, the
Huang Ziwei
Lu Zhixian
Shan Simei
Wang Jialun
Zhang Zhijia
Audet Maury
Drinker Biddle & Reath LLP
Tate Christopher R.
Thomas Jefferson University
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