Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-03-21
2003-04-08
Nguyen, Dave T. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S455000, C435S375000, C435S252300, C435S419000, C435S325000
Reexamination Certificate
active
06544956
ABSTRACT:
INTRODUCTION
1. Field of the Invention
The invention relates to a class of peptides which disrupts mitotic function in cells.
2. Background of the Invention
Early seed development in angiosperms is characterized by rapid cell division and differentiation followed by cessation of cell division and the start of cell expansion during which DNA endoreduplication and massive synthesis of storage proteins, carbohydrates and lipids occur in the endosperm of cereals and in the cotyledons of legumes (1). The molecular mechanisms underlying these early events are poorly understood. The temporal expression of 2S albumins, a diverse group of water soluble proteins found in developing seeds, has been shown to coincide with the initiation of cell expansion after the cells stopped dividing in developing pea embryos (2). In situ hybridization work on Arabidopsis involving actively dividing and nondividing parynchema cells also suggests the possible existence of a regulatory mechanism that is common to 2S albumin gene expression and mitotic activity (3). However, the functional role of 2S albumins in regulation of mitosis during seed development remains to be established.
SUMMARY OF THE INVENTION
The invention provides methods and compositions of selectively modulating mitotic function in a target cell demonstrating undesirable mitotic function. Suitable target cells include mammalian, plant and bacterial cells, which cells may be in vitro or in situ. The general methods involve introducing into the target cell an effective amount of a modulator of mitotic function comprising a Gm2S-1 peptide, and/or contiguous acidic amino acids, such as Asp or Glu, whereby the undesirable mitotic function of the cell is selectively modulated. In particular embodiments, the Gm2S-1 peptide comprises the contiguous acidic amino acids and/or comprises residues 33-43, residues 85-91, residues 123-127, residues 85-127, at least 12 contiguous residues of SEQ ID NO:2, residues 1-35 and/or SEQ ID NO:2. The peptide may be introduced by transfecting the cell with a nucleic acid which encodes the peptide or comprises SEQ ID NO:1 or a fragment thereof which modulates the expression of a resident Gm2s-1 peptide-encoding gene or transcript.
The invention encompasses a variety of compositions which may be used in the subject methods including modulators of mitotic function, Gm2S-1 peptides, Gm2S-1 peptide-encoding nucleic acids, Gm2S-1 peptide-specific binding agents such as antibodies, and nucleic acid hybridization probes and primers comprising a strand of SEQ ID NO:1 or a fragment thereof sufficient to specifically hybridize with and thereby facilitate identifying, cloning, amplifying and/or modulating the expression of a Gm2S-1 peptide-encoding gene.
DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION
The invention provides methods and compositions of selectively disrupting mitotic function in a target cell demonstrating undesirable mitotic function. Hence, the methods may be used to interfere with (e.g. promote, prevent or delay) targeted cell division. The invention is applicable to a wide variety of indications where mitotic function is undesirable quantitatively, qualitatively, spacially, temporally, etc. For example, in one particular embodiment, the methods and compositions are used to control undesirable growth of cells in human neoplasia, such as cancer, restinosis, etc. In another embodiment, the invention is used to prevent the normal division of harmful micororganisms, e.g. pathogenic bacteria such as described in Medical Microbiology 4
th
Ed. (S. Baron, Ed., 1996, UT Med Branch at Galveston). In yet another embodiment, the invention is used to regulate plant seed development by controling the timing of the termination of cell division that allows DNA endoreduplication to occur.
The general methods involve introducing into the target cell an effective amount of a peptide modulator, preferably a disruptor, of mitotic function comprising contiguous acidic amino acids, preferably at least 6, more preferably at least 7, more preferably at least 8 such as Asp or Glu, sufficient to selectively modulate mitotic function of a cell. As demonstrated herein, the invention encompasses a wide variety of suitable methods, amounts, and peptide lengths and compositions, which are readily optimized empirically.
In particular embodiments, the modulator peptide comprises a Gm2S-1 peptide which comprises at least 5, preferably at least 6, more preferably at least 7 of the contiguous acidic amino acids. In other preferred embodiments, the peptide comprise a Gm2S-1 peptide comprising at least an 8, preferably at least a 10, more preferably at least a 20 residue domain of SEQ ID NO:2 sufficient to selectively modulate mitotic function of the cell. Such peptides may derive from the Gm2s-1 signal peptide (residues 01-21 of SEQ ID NO:2), lunasin (residues 22-64 of SEQ ID NO:2), the Gm2S-1 linker peptide (residues 65-81 of SEQ ID NO:2) or alisin (residues SEQ ID NO:2, residues 82-158). In other preferred embodiments, the peptide comprises residues 33-43 of SEQ ID NO:2, residues 85-91 of SEQ ID NO:2, residues 123-127 of SEQ ID NO:2, residues 85-127 of SEQ ID NO:2 and/or at least 12 contiguous residues of SEQ ID NO:2, residues 1-35. The contiguous acidic amino acids are preferably proximate to (i.e. within 12 residues, preferably within 6 residues, more preferably within 3 residues) or at the C-terminus of the modulator. The modulator may comprise a wide variety of additional moieties, especially moities positioned N-terminally relative to the acidic amino acids, including moieties which provide for detection, targeting, stability, proteolytic resistance, etc.
The subject modulators comprising Gm2S-1 peptides provide Gm2S-1 peptide specific activity or function, such as Gm2S-1-specific disruption of mitotic function, ligand/antibody binding or binding inhibitory, immunogenicity, etc. Gm2S-1 peptide-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in,animals (e.g. gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the molecular interaction of a Gm2S-1 peptide with a binding target is evaluated. The binding target may be a natural intracellular binding target such as a Gm2S-1 peptide regulating protein (e.g. mapmodulin, Ulitzur et al., 1997, PNAS 94, 5084-5089) or other regulator that directly modulates a Gm2S-1 peptide activity or its localization; or non-natural binding target such a specific immune protein such as an antibody, or an Gm2S-1 peptide specific agent such as those identified in bio/chemical screening assays. Gm2S-1 peptide-binding specificity may assayed by mitotic disruption assays described below, binding equilibrium constants (usually at least about 10
7
M
−1
, preferably at least about 10
8
M
−1
, more preferably at least about 10
9
M
−1
), by the ability of the subject peptides to function as negative mutants in a Gm2S-1 peptide-expressing cells, to elicit a Gm2S-1 peptide specific antibody in a heterologous host (e.g. a rodent or rabbit), etc. The Gm2S-1 peptide binding specificity of preferred Gm2S-1 peptides necessarily distinguishes that of the tubulin, MAPs and Mapmodulin.
In particular embodiments, modulators comprising Gm2S-1 peptides are isolated or pure: an “isolated” peptide is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, and more preferably at least about 5% by weight of the total peptide in a given sample and a pure peptide constitutes at least about 90%, and preferably at least about 99% by weight of the total peptide in a given sample. The peptides may be synthesized, produced by recombinant technology, or purified from cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold
de Lumen Benito O.
Galvez Alfredo F.
Nguyen Dave T.
Osman Richard Aron
The Regents of the University of California
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