Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-10-19
2002-07-16
Ponnaluri, Padmashri (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S007100, C435S069100, C435S235100, C435S091500, C435S091500, C435S091500, C435S091500
Reexamination Certificate
active
06420110
ABSTRACT:
BACKGROUND OF THE INVENTION
High throughput screening has become a dominant tool in the pharmaceutical industry for the discovery of lead compounds that can be modified into candidates for drug development. For instance, it is abundantly used for identification of ligands with high affinity for receptors. In this regard, combinatorial techniques have provided approaches to generating and deconvoluting large libraries of test compounds in high throughput screens. It involves selection and amplification of a subset of molecules with desired biological properties from complex libraries.
One technique which has emerged for identification of peptide leads involves the use of peptide display methodologies such as phage display. Phage-displayed peptide libraries can comprise vast collections of short, randomized polypeptides that are displayed on the surface of a filamentous bacteriophage particle. Thus, each “peptide” is actually the N-terminal sequence of a phage-coat protein, that is encoded by a randomly-mutated region of the phage genome responsible for the production of the coat protein. In this manner, each unique peptide in the library is physically linked with the DNA molecule encoding it. Antibodies and other binding molecules can be used as “targets” to specifically select rare phage clones bearing ligand peptides, and sequencing of the corresponding viral DNA will reveal their amino acid sequences. Relatively high-affinity peptides for a variety of peptide- and non-peptide-binding targets have been affinity-isolated from epitope libraries. This technology has been used to map epitopes on proteins and to find peptide mimics for a variety of target molecules. Many powerful applications can be envisioned in the areas of drug design and the development of diagnostic markers, vaccines and toleragens.
For the purposes of drug discovery, there are potential advantages in the use of genetically encoded libraries, such as phage display (Scott et al,
Science
249, 386 (1990); Devlin et al.,
Science
249, 386 (1990)), “peptide on plasmid” (Cull et al.
PNAS
89, 1865 (1992)), and in vitro translation-based systems (Mattheakis et al.
PNAS
91, 9022 (1994)), compared to the use of synthetic small molecule libraries (Bunin et al.
PNAS
91, 4708 (1994); Gordon et al.
J. Med. Chem.
37, 1385 (1994); and Dooley et al.,
Science
266, 2019 (1994)). The genetic encoding of libraries allows the resynthesis and rescreening of molecules with a desired binding activity. The resulting amplification of interacting molecules in subsequent rounds of selection can lead to the isolation of extremely rare, specific binders from a large pool of molecules.
However, despite the success of these methods, they suffer from numerous sources of error and bias, such as very low initial concentrations of species, non-specific binding, and, significantly, the sampling of only a fraction of the library at the end of an experiment.
SUMMARY OF THE INVENTION
One aspect of the invention provides a method for generating a peptide with a selected biological activity, comprising the steps of:
(i) providing a peptide display library comprising a variegated population of test peptides expressed on the surface of a population of display packages;
(ii) in a display mode, isolating, from the peptide display libary, a sub-population of display packages enriched for test peptides which have a desired binding specificity and/or affinity for a cell or a component thereof;
(iii) in a secretion mode, simultaneously expressing the enriched test peptide sub-population under conditions wherein the test peptides are secreted and are free of the display packages; and
(iv) assessing the ability of the secreted test peptides to regulate a biological process in a target cell.
For instance, the peptide display library can be a phage display library, e.g., which utilizes phage particles such as M13, f1, fd, If1, Ike, Xf, Pf1, Pf3, &lgr;, T4, T7, P2, P4, &phgr;X-174, MS2 or f2. In preferred embodiments, the phage display library is generated with a filamentous bacteriophage specific for
Escherichia coli
and the phage coat protein is coat protein III or coat protein VIII. For instance, the filamentous bacteriophage can be M13, fd, and f1.
In other embodiments, the peptide display library is a bacterial cell-surface display library or a spore display library.
In certain embodiments, the test peptides are enriched from the peptide display library in the display mode by a differential binding means comprising affinity separation of test peptides which specifically bind the cell or component thereof from test peptides which do not. For example, the differential binding means can include panning the peptide display library on whole cells, affinity chromatographic means in which a component of a cell is provided as part of an insoluble matrix (.e.g, a cell surface protein attached to a polymeric support), and/or immunoprecipitating the display packages.
In the display mode, the test peptides can be enriched for those which bind to a cell-type specific marker and/or a cell surface receptor protein. For example, the test peptide library can be enriched in the display mode for test peptides which bind to a G-protein coupled receptor, such as a chemoattractant peptide receptor, a neuropeptide receptor, a light receptor, a neurotransmitter receptor, a cyclic AMP receptor, or a polypeptide hormone receptor. In other embodiments, the test peptide library can be enriched in the display mode for test peptides which bind to a receptor tyrosine kinase, such as an EPH receptor. In still other embodiments, the test peptide library can be enriched in the display mode for test peptides which bind to a cytokine receptor or an MIRR receptor. In certain embodiments, the test peptide library can be enriched in the display mode for test peptides which bind to an orphan receptor.
In preferred embodiments, the peptide display library includes at least 10
3
different test peptides.
In preferred embodiments, the test peptides are 4-20 amino acid residues in length.
In certain embodiments, each of the test peptides are encoded by a chimeric gene comprising (i) a coding sequence for the test peptide, (ii) a coding sequence for a surface protein of the display package for displaying the test peptides on the surface of a population of display packages, and (iii) RNA splice sites flanking the coding sequence for the surface protein, wherein, in the display mode, the chimeric gene is expressed as fusion protein including the test peptide and the surface protein, whereas in the secretion mode, the test peptide is expressed without the surface protein as a result of the coding sequence for the surface protein being removed by RNA splicing.
In preferred embodiments, the test peptides are expressed by a eukaryotic cell, more preferably a mammalian cell, in the secretion mode.
In preferred embodiments, the target cell is a eukaryotic cell, more preferably a mammalian cell such as a human cell.
In certain embodiments, the biological process scored for in the secretion mode includes a change in cell proliferation, cell differentiation or cell death. In other embodiments, the biological process which is detected is changes in intracellular calcium mobilization, intracellular protein phosphorylation, phospholipid metabolism, and/or expression of cell-specific marker genes.
In certain embodiments, the target cell includes a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the signal transduction acitivity of the cell surface receptor protein, expression of the reporter gene providing the detectable signal. For instance, the reporter gene can encode a gene product that gives rise to a detectable signal selected from the group consisting of: color, fluorescence, luminescence, cell viability relief of a cell nutritional requirement, cell growth, and drug resistance. In preferred embodiments, the reporter gene encodes a gene product selected from the group consisting of chloramphenicol acetyl t
Gyuris Jeno
Morris Aaron J.
GPC Biotech Inc.
Halstead David P.
Ponnaluri Padmashri
Ropes & Gray
Vincent Matthew P.
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