Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1996-11-13
2001-02-13
Spector, Lorraine (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007800, C435S173300, C435S069100, C435S325000, C435S358000, C435S360000, C435S364000, C435S365000, C435S365100, C435S252300, C435S320100, C530S350000, C536S023500
Reexamination Certificate
active
06187550
ABSTRACT:
FIELD OF THE INVENTION
This invention features methods and non-naturally occurring cells for screening compositions and genes which interact with interleukin 1&bgr; and interleukin-1 beta converting enzyme (ICE) processing and/or activation.
BACKGROUND OF THE INVENTION
Interleukin-1 (IL-1) plays an important role in the pathogenesis of several inflammatory disorders. Two forms of IL-1 proteins have been described, interleukin-1-alpha (IL-1&agr;) and interleukin-1-beta (IL-1&bgr;). This application will use the terms IL-1&agr; and IL-1&bgr; to denote the respective forms of IL-1 proteins consistent with the usage of such terms in the scientific literature. See: Young et al. “Human Interleukin Ib is not secreted from Hamster Fibroblasts when expressed constitutively from transferred cDNA”,
Journal of Cell Biology
, Vol. 107, 447-455 (1988). Both forms of IL-1 are synthesized as approximately 31 kDa precursor molecules that are subsequently processed to generate 17 kDa mature molecules. Although both forms of IL-1 are secreted proteins, each protein lacks signal peptides. The mechanism of the secretion has not been fully defined.
IL-1&agr; and IL-1&bgr; are products of distinct genes. The proteins share only 27-33% of their amino acids even though each protein has a similar biological activity and interact with the same receptor. IL-1&agr; precursor (preIL-1&agr;) is almost as active as the mature form. In contrast, IL-1&bgr; precursor (preIL-1&bgr;) has no biological activity until further processed to a mature form. This application will use the term mature, bioactive IL-1&bgr; (matIL-1&bgr;) to highlight and emphasize that a precursor composition has been processed to the mature, active composition. In humans, IL-1&bgr; is the predominant species and may play a more important role in certain disease states.
Only certain cell types process preIL-1&bgr; and secrete matIL-1&bgr;. Monocytes and macrophages are the most efficient producers and secretors of IL-1&bgr;. Of the two forms of IL-1 synthesized and secreted following activation of monocytes and macrophages, IL-1&bgr; is the most abundant form.
The cellular processing of preIL-1&bgr; to mature, bioactive IL-1&bgr; is mediated by the enzyme ICE, a cysteine protease. ICE is synthesized as a 45 kDa precursor molecule which is processed in vivo to form fragments of 20 and 10 kDa. These two fragments are combined or folded, in vivo, to form the active enzyme.
IL-1&bgr; plays a critical role in the pathogenesis of several inflammatory, autoimmune, and leukemic disease states. Thus, an understanding of the release and processing of IL-1&bgr; and its precursors is desired. The present knowledge is, in part, limited by a lack of models on the cellular level for the making and processing of IL-1&bgr;. In vivo, at the cellular level, monocytes and macrophages produce small quantities of IL-1&bgr;, for only a limited time. It is difficult to grow monocytes and macrophages for long periods of time. There are large differences and variations between freshly prepared monocytes and macrophages. In naturally occuring cells, it is difficult to study preIL-1&bgr; and ICE interactions. As a consequence, the role of various regions or domains of preIL-1&bgr; and ICE in the processing, activation and release of ICE and IL-1&bgr;, and the structural significance of various amino acid residues remain unclear.
A consistent model capable of alteration and manipulation is desired to further the understanding of the release and processing of IL-1&bgr; and ICE and as a screen for compositions which may interact with the processing and release of IL-1&bgr; and ICE. A consistent model that mimics the induction of preIL-1&bgr; upon the application of a stimulus, and consistently expresses ICE, is desired.
Compositions which alter the production, processing and release of IL-1&bgr; and ICE have interest as therapeutics to modulate the inflammation response. IL-1&bgr; and ICE themselves may also have utility to modulate inflammatory responses.
SUMMARY OF THE INVENTION
The present invention is directed to methods and cell lines for screening compositions and genes for ICE activity or ICE inhibitory or agonist activity. One embodiment of the present invention features a non-naturally occurring cell which cell stably incorporates a gene for preIL-1&bgr; operably linked to a promoter. The cell produces preIL-1&bgr;, but, in the absence of ICE, is unable to process preIL-1&bgr; to make mature, bioactive IL-1&bgr;. A cell having a gene for preIL-1&bgr; operably linked to a promoter has utility to identify nucleic acids coding ICE or ICE-like compositions and to identify ICE-like compositions which can be applied directly to the cell.
The term “non-naturally occurring” refers to an object which has been manipulated or changed from its natural state. As applied to a cell, a non-naturally occurring cell has a non-naturally occurring nucleic acid, or makes a non-naturally occurring peptide, or is fused to a cell to which it is not combined with in nature. The term “non-naturally occurring nucleic acid” refers to a portion of a genomic nucleic acid, cDNA, semisynthetic nucleic acid, or synthetic original nucleic acid which, by virtue of its origin or manipulation, is not associated with all of a nucleic acid to which it is associated in nature, or is linked to a nucleic acid or other chemical agent other than that to which it is linked in nature, or does not occur in nature. The term “non-naturally occurring peptide” refers to a portion of a larger naturally occurring peptide or protein, or semisynthetic or synthetic peptide, which by virtue of its origin or manipulation, is not associated with all of the peptide to which it is associated in nature, or is linked to a peptide, functional group or chemical agent other than that to which it is linked in nature, or does not occur in nature.
As used herein, the term “operably linked” refers to a nucleic acid which is associated in a manner to effect transcription and translation by a cell in which it is placed.
The term “stably incorporated” refers to maintaining a feature or gene through several cell divisions.
The term “unable to process preIL-1&bgr;” refers to a substantial absence of mature, bioactive IL-1&bgr; in the presence of preIL-1&bgr;. A substantial absence of mature bioactive IL-1&bgr; is less than 5-10% of total preIL-1&bgr; protein.
As used herein, “ICE” refers to IL-1&bgr; converting enzyme as defined in European Patent Application No. 92307479.3 having a filing date of Aug. 14, 1992, by Merck & Company, Inc. ICE-like compositions refers to molecules and compositions which have ICE activity, that is, which function to convert the 31 kDa precursor protein, preIL-1&bgr;, into a 17 kDa mature bioactive IL-1&bgr; molecule. By way of example, without limitation, such ICE-like molecules comprise separately expressed 10 and 20 kDa fragments, recombinant ICE, fragments derived from the 45 kDa preICE molecule (including but not limited to a 32 kDa fragment) and proteins resembling ICE with non-critical amino acid substitutions, deletions and additions. Separately expressed fragments and recombinant ICE further comprise such additional amino acids which are added to mammalian proteins when nucleic acid coding such proteins is expressed in bacterial systems and restriction sites and other features, which permit cloning.
Preferably, the cell has a gene for preIL-1&bgr; stably incorporated within its genome. Such cell is a model cell for a cell which does not process and secrete preIL-1&bgr;. A preferred cell line comprising cells designated COS pre 11 has been deposited with the American Type Culture Collection (ATCC) of Rockville, Md. on Jul. 27, 1994 under the terms of the Budapest Treaty and in accordance with U.S. Patent Practice. This cell-line has the ATCC designation of Accession No. CRL 11693.
One embodiment of the present invention comprises the step of applying preICE, ICE, ICE-like compositions and other compositions which are to be evaluated for activity in forming mature, bioactive IL-1&bgr; to a cell ha
Ghayur Tariq
McGuinness Lorraine M.
BASF AG
DeConti, Jr. Giulio A.
Hanley Elizabeth A.
Lahive & Cockfield LLP
Spector Lorraine
LandOfFree
Methods and cell lines for screening compositions and genes... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods and cell lines for screening compositions and genes..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods and cell lines for screening compositions and genes... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2569656