Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-08-15
2004-05-18
Ungar, Susan (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S351000, C530S387300, C530S387700, C530S388100, C530S388800, C435S069100, C435S069500, C435S069700, C435S071300, C424S134100, C424S138100, C424S141100, C424S236100, C424S238100
Reexamination Certificate
active
06737511
ABSTRACT:
FIELD
This invention relates to modification of the apoptotic response of target cells, for instance target cells in a subject. More specifically, it relates to apoptosis-modifying fusion proteins with at least two domains, one of which targets the fusion protein to a target cell, and another of which modifies an apoptotic response of the target cell.
BACKGROUND
Tissue and cell homeostasis in multicellular organisms is largely influenced by apoptosis, the phenomenon of programmed cell death by which an intra- or extra-cellular trigger causes a cell to activate a biochemical “suicide” pathway. Morphological indicia of apoptosis include membrane blebbing, chromatin condensation and fragmentation, and formation of apoptotic bodies, all of which take place relatively early in the process of programmed cell death. Degradation of genomic DNA during apoptosis results in formation of characteristic, nucleosome sized DNA fragments; this degradation produces a diagnostic ~180 bp laddering pattern when analyzed by gel electrophoresis. A later step in the apoptotic process is degradation of the plasma membrane, rendering apoptotic cells leaky to various dyes (e.g., trypan blue and propidium iodide). Apoptotic cells are usually engulfed and destroyed early in the death process; thus, apoptosis tends not to be associated with inflammation caused by cytoplasm leakage, as is found in necrosis.
Various in vivo triggers can induce apoptosis; the paradigmatic trigger is a shortage of one or more necessary growth factors. Apoptosis plays a significant role in development of the neural system (reviewed in Cowan et al.,
Science
225:1258-1265, 1984; Davies,
Development
101:185-208, 1987; Oppenheim,
Annu. Rev. Neurosci
. 14:453-501, 1991) and lymphoid system (reviewed in Blackman et al.,
Science
248:1335-1341, 1990; Rothenberg,
Adv. Immunol
. 51:85-214, 1992) of vertebrates. System development occurs through selective apoptotic extinction of certain cell populations.
In spite of much study, the molecular mechanisms of apoptosis are not fully elucidated. It does appear, however, that different apoptosis inducers may trigger different apoptotic pathways. For instance, certain pathways are transcription-dependent, in that apoptosis requires the synthesis of new proteins after stimulation by, for instance, withdrawal of growth factors. Staurosporine, a non-specific kinase inhibiter, in contrast, stimulates a transcription-independent pathway. Transcription dependent and independent pathways appear to share downstream components, including the ICE family of proteases (caspases). See Rubin,
British Med. Bulle
., 53:617-631, 1997, for a review of apoptosis in neurons; More general reviews include Ashkenazi and Dixit,
Science
281:1305-1308; Thornberry and Lazebnik,
Science
281:1312-1316; and Adams and Cory,
Science
281:1322-1326.
Apoptosis is recognized as a gene-directed event, controlled by a complex set of interacting gene products that inhibit or enhance apoptosis (Williams and Smith,
Cell
74:777-779, 1993; reviewed in White,
Genes Dev
. 10:1-15, 1996). Extensive effort is currently underway to identify and characterize the genes involved in this process. The first protein characterized as influencing apoptosis was Bcl-2 (Cleary et al.,
Cell
47:19-28, 1986; Tsujimoto and Croce,
Proc. Natl. Acad Sci. USA
83:5214-5218, 1986). Since its discovery, several Bcl-2-related proteins (the Bcl-2 family of proteins) have been identified as being involved in regulation of apoptosis (White,
Genes Dev
. 10:1-15, 1996; Yang et al.,
Cell
80:285-291, 1995). One such is Bcl-x, which is expressed in two different forms, long (Bcl-x
L
) and short (Bcl-x
S
) (Boise et al.,
Cell
74:597-608, 1993).
Bcl-x
L
and certain other members of the Bcl-2 family are, like Bcl-2 itself, powerful inhibitors of cell death (the “anti-death” Bcl-2 family members). Genetic overexpression of Bcl-2 has been shown to block apoptosis in the nervous system of transgenic mice (Chen et al.,
Nature
385:434-439, 1997; Henkart,
Immunity
4:195-201, 1996; Lippincott-Schwartz et al.,
Cell
67:601-616, 1991; Hunziker et al.,
Cell
67:617-627, 1991; Krajewski et al.,
Cancer Research
53:4701-4714, 1993; Martinou et al.,
Neuron
13:1017-1030, 1994).
Other members of the Bcl-2 protein family, including Bcl-x
S
, Bad and Bax, are potent enhancers of apoptosis and therefore toxic to cells (“pro-death” Bcl-2 family members). Though the mechanism of apoptosis induction by these proteins remains unknown, it has been suggested that Bad binding to Bcl-x
L
may promote cell death (Yang et al.,
Cell
80:285-291, 1995; Zha et al.,
J Biol. Chem
272:24101-24104, 1997) and that phosphorylation of Bad may prevent its binding to Bcl-x
L
, thereby blocking cell death (Zha et al.,
J Biol. Chem
. 272:24101-24104, 1997; Zha et al.,
Cell
87:619-628, 1996).
In addition to its involvement in neuronal and lymphoid system development and overall cell population homeostasis, apoptosis also plays a substantial role in cell death that occurs in conjunction with various disease and injury conditions. For instance, apoptosis is involved in the damage caused by neurodegenerative disorders, including Alziheimer's disease (Barinaga,
Science
281:1303-1304), Huntington's disease, and spinal-muscular atrophy. There is also a substantial apoptotic component to the neuronal damage caused during stroke episodes (reviewed in Rubin,
British Med. Bulle
., 53(3):617-631, 1997; and Barinaga,
Science
281:1302-1303), and transient ischemic neuronal injury, as in spinal cord injury. It would be of great benefit to prevent undesired apoptosis in various disease and injury situations.
Treatment with standard apoptosis inhibitory molecules, for instance peptide-type caspase inhibitors (e.g., DEVD-type), though useful for laboratory experiments where microinjection can be employed, has proven unsatisfactory for clinical work due to low membrane permeability of these inhibitors. Transfection of cells with various native proteins, including members of the Bcl-2 family of regulatory proteins, has dual disadvantages. First, transfection is usually not cell-specific, and thus may disrupt apoptotic processes non-specifically in all cells. Second, transfection tends to provide long term alterations in the apoptotic process, in that once a transgene is integrated and functional in the genome of target cells, it may be difficult to turn off. Especially in instances of stroke episodes or transient ischemic neuronal injury, it would be more advantageous to be able to apply apoptosis regulation for short periods of time. Therefore, there is still a strong need to develop pharmaceutical agents that overcome these disadvantages.
Cancer and other hyper-proliferative cell conditions can be viewed as inappropriate escape from appropriate cell death. As such, it would be advantageous to be able to enhance apoptosis in certain of these cells to stop unregulated or undesired growth. Various attempts have been made to selectively eliminate cancerous cells through the use of targeted immunotoxins (genetic or biochemical fusions between a toxic molecule, for instance a bacterial toxin, and a targeting domain derived, typically from an antibody molecule).
One bacterial toxin that has been employed in attempts to kill cancerous cells is diphtheria toxin (DT). Diphtheria toxin has three structurally and functionally distinct domains: (1) a cell surface receptor binding domain (DTR), (2) a translocation domain (DTT) that allows passage of the active domain across the cell membrane, and (3) the A (enzymatically active) chain that, upon delivery to a cell, ADP-ribosylates elongation factor 2 and thereby inactivates translation. Altering the receptor specificity of the diphtheria toxin has been used to generate toxins that may selectively kill cancer cells in vitro (Thorpe et al.,
Nature
271:752-755, 1978) and in man (Laske et al.,
Nature Medicine
3:1362-1368, 1997). Promising though they might have seemed, these and similar hybrid immunotoxins have proven to be substantially l
Collier R. John
Xiuhuai Liu
Youle Richard J.
Davis Minh Tam
Klarquist & Sparkman, LLP
The United States of America as represented by the Department of
Ungar Susan
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