Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1998-11-17
2003-07-08
Ponnaluri, Padmashri (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S069100, C435S069800, C435S320100, C435S091500, C435S091500, C435S091500, C435S091500, C435S091500, C536S023100, C536S023400, C536S025320
Reexamination Certificate
active
06589730
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to screening for protein-protein interactions, and in particular, to identification of specific protein-protein interactions which lead to internalization of the protein-protein complex and transgene expression in the target cell.
BACKGROUND OF THE INVENTION
Bacteriophage expressing a peptide on its surface has been used to identify protein binding domains, including antigenic determinants, antibodies that are specifically reactive, mutants with high affinity binding, identify novel ligands, and substrate sites for enzymes. In its most common form, a peptide is expressed as a fusion protein with a capsid protein of a filamentous phage. This results in the display of the foreign protein on the surface of the phage particle. Libraries of phages are generated that express a multitude of foreign proteins. These libraries are bound to a substrate or cell that presents the binding partner of interest. This screening process is essentially an affinity purification. Bound phage are recovered, propagated, and the gene encoding the foreign protein may be isolated and characterized. This technology is commonly referred to as “phage display.”
Through a process called “biopanning,” the specific phage carrying a peptide or protein that interacts with a protein or other moiety on a solid phase can be identified and isolated. However, in some applications, binding or binding affinity is not the sole critical parameter. For example, in gene therapy, a gene sequence needs to be introduced into a cell. In preferred methods, the gene sequence is targeted to particular cells by way of a ligand/cell surface receptor interaction. Thus, the ligand must not only bind to the cells but must also be internalized. A native ligand that is internalized, when used in a system for gene therapy may not be efficiently internalized. For example, both FGF2 and EGF are internalizing ligands; however, of these two ligands, FGF (or polypeptides reactive with the FGF receptor) is currently preferred as a gene targeting ligand.
Phage libraries can be screened for internalizing ligands by biopanning on live cells and rescuing internalized phage from the cells after stripping off externally bound phage (e.g., acid elution). However, this method may result in recovery of undesired phage that bind very tightly or are only partially internalized. Moreover, phage that are internalized and subjected to proteases lose infectivity and can not be recovered. Accordingly, current methodologies are inadequate to determine the usefulness of ligands for gene therapy.
Further, identification of target cells or tissues that are able to internalize ligands and express a transgene would readily allow one to identify specific target cells for known or putative ligands as well as allow one to identify ligands for specific cell or tissue types. However, current methods of target cell identification are hampered by the same difficulties, as noted above, with regard to screening for internalizing ligands. Accordingly, current methodologies are inadequate to determine which cell or tissue types are useful targets for ligand mediated gene therapy.
Thus, current screening methods are inadequate for identifying cell or tissue types that bind and internalize known or putative ligands. The present invention discloses a ligand display method that identifies target cells or tissue types that bind internalizing ligands as well as identifying the specific ligands that internalize in the target cell or tissue, and further provides other related advantages.
SUMMARY OF THE INVENTION
Within one aspect of the present invention, a method of selecting internalizing ligand/anti-ligand pairs is presented, comprising: (a) contacting a ligand displaying genetic package(s) with a cell(s), wherein the package carries a gene encoding a detectable product which is expressed upon internalization of the package; and (b) detecting product expressed by the cell(s); thereby selecting ligand/anti-ligand pairs.
In another aspect, the invention provides a method of identifying a ligand or anti-ligand of an internalizing ligand/anti-ligand pair, comprising: (a) contacting one or more ligand displaying genetic packages with a cell(s), wherein each package carries a gene encoding a detectable product which is expressed upon internalization of the package, and wherein the cell(s) expresses an anti-ligand-receptor fusion protein on its surface; (b) detecting product expressed by the cell(s); and (c) recovering a nucleic acid molecule encoding an internalizing ligand and/or a nucleic acid molecule encoding an internalizing anti-ligand from the cell(s) expressing the product, and thereby identifying a ligand or anti-ligand of a internalizing ligand/anti-ligand pair.
In yet another aspect, the invention provides a method of identifying a ligand or anti-ligand of an internalizing ligand/anti-ligand pair, comprising: (a) contacting one or more ligand displaying genetic packages with a cell(s), wherein each package carries a gene encoding a detectable product which is expressed upon internalization of the package, and wherein the cell(s) expresses an anti-ligand-receptor fusion protein on its surface; (b) incubating the cell(s) under selective conditions; and (c) recovering a nucleic acid molecule encoding an internalizing ligand and/or a nucleic acid molecule encoding an internalizing anti-ligand from the cell(s) which grow under the selective conditions; thereby identifying a ligand or anti-ligand of a internalizing ligand/anti-ligand pair, comprising.
In yet another aspect, a method is provided for a high throughput method of identifying a ligand or anti-ligand of an internalizing ligand/anti-ligand interactions, comprising: (a) contacting one or more ligand displaying genetic packages with a cell(s) in an array, wherein each package carries a gene encoding at least one detectable product which is expressed upon internalization of the package; and (b) detecting product(s) expressed by the cell(s) in the array, and thereby identifying a ligand or anti-ligand of a internalizing ligand/anti-ligand interactions. In one embodiment, the array contains cells expressing a library of anti-ligand-receptor fusion proteins. In another embodiment, the ligand displaying package comprises a library of ligand displaying packages.
In preferred embodiments, the ligand displaying genetic package comprises a bacteriophage. The bacteriophage are filamentous phage or lambdoid phage in other preferred embodiments. In some embodiments, the bacteriophage carries a genome vector.
In other embodiments, the library is a cDNA library, an antibody gene library, a random peptide gene library, or a mutein library. In other preferred embodiments, the detectable product is selected from the group consisting of green fluorescent protein, &bgr;-galactosidase, secreted alkaline phosphatase, chloramphenicol acetyltransferase, luciferase, human growth hormone and neomycin phosphotransferase.
In other embodiments, the cells may be isolated by flow cytometry, for example. In further embodiments, the methods further comprise recovering a nucleic acid molecule encoding the ligand and/or a nucleic acid molecule encoding the anti-ligand from the cell(s) expressing the product.
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Larocca et al., “Targeted Transduction of Mammalian Cells Using a FGF2 Modified Filamentous Bacteriophage,”Cancer Gene Therapy 4(6): Abstract No. O-46, p. S24, 1997.
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Larocca et al., “T
Baird Andrew
Kassner Paul
Larocca David
Ponnaluri Padmashri
Seed Intellectual Property Law Group PLLC
Selective Genetics Inc.
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