Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-02-26
2002-09-17
Ponnaluri, Padmashri (Department: 1618)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S235100, C435S320100, C435S091500, C435S091500, C435S091500, C435S091500, C435S091500, C435S091500, C536S023100
Reexamination Certificate
active
06451527
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to genetic package display (e.g., phage display), and in particular, to selection of ligands that bind to a cell surface receptor and internalize.
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 many 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 I 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.
Phage libraries can be screened for potentially internalizing ligands by biopanning on live cells and rescuing internalized phage from the cells after stripping off externally bound phage (erg., 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 selecting peptide or protein ligands that bind to a cell surface receptor and internalize. The present invention discloses a display methods that select peptide or protein ligands that internalize, and further provides other related advantages.
SUMMARY OF THE INVENTION
Within one aspect of the present invention, a method of selecting internalizing ligands displayed on a genetic package 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 internalizing ligands displayed on a genetic package.
In another aspect, the invention provides a method of identifying an internalizing ligand displayed on a genetic package, 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, (b) detecting product expressed by the cell(s); and (c) recovering a nucleic acid molecule encoding an internalizing ligand from the cell(s) expressing the product, and thereby identifying an internalizing ligand displayed on a genetic package.
In yet another aspect, the invention provides a method of identifying an internalizing ligand displayed on a genetic package, comprising: (a) contacting one or more ligand displaying genetic packages with a cell(s), wherein each package carries a gene encoding a selectable product which is expressed upon internalization of the package, (b) incubating the cell(s) under selective conditions; and (c) recovering a nucleic acid molecule encoding an internalizing ligand from the cell(s) which grow under the selective conditions; thereby identifying an internalizing ligand displayed on a genetic package.
In yet another aspect, a method is provided for a high throughput method of identifying an internalizing ligand displayed on a genetic package, 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 an internalizing ligand displayed on a genetic package. In one embodiment, the ligand displaying package comprises a library of ligand displaying packages.
In another aspect, the present invention provides a method of identifying an internalizing ligand displayed on a genetic package, comprising: (a) contacting one or more ligand displaying a genetic packages with a cell(s), wherein each package carries a selectable marker which is detectable upon internalization of the package, (b) detecting the selectable marker internalized by the cells; and (c) recovering a nucleic acid molecule encoding an internalizing ligand from the cell(s) carrying the selectable marker, thereby identifying an internalizing ligand displayed on a genetic package.
In related embodiments, the selectable marker is selected from reporter gene expression, expression of a gene that confers the ability to permit cell growth under selection conditions, non-endogenous nucleic acid sequences that permit PCR amplification, and nucleic acid sequences that can be purified by protein/DNA binding.
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 bacteriophage carries a hybrid 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 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.
Larocca et al., “Targeted Gene Delivery to Mammalian Cells Via Fibroblast Growth Factor (FGF-2) Display Phage,”Cancer Gene Therapy 5(6): Abstract No. PD-31, p.
Baird Andrew
Kassner Paul
Larocca David
Ponnaluri Padmashri
Seed Intellectual Property Law Group PLLC
Selective Genetics Inc.
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