Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-24
2004-04-20
Ponnaluri, Padmashri (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S069100, C435S320100, C435S091500, C435S091500, C435S091500, C435S091500, C435S091500, C536S023100
Reexamination Certificate
active
06723512
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 useful in gene therapy, screening, and in various methodologies. The methods described herein are also referred to as Ligand Identification Via Expression or “LIVE™”.
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 coat 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/cell surface receptor interaction. Thus, the ligand must not only bind to the cells but must also be internalized and lead to expression of the introduced nucleic acid sequence. A native ligand that is internalized, when used in a system for gene therapy may not be efficiently internalized or while internalized may not lead to gene expression. For example, both FGF2 and EGF are internalizing ligands. Further, while many ligands can be found to internalize many do not facilitate tranduction of the targeted cell, leaving the internalized nucleic acid sequence in a non-functional state.
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 (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.
Generally speaking, the selection of ligands from phage display libraries or other genetic packages relies on peptide affinity and avidity. The number of phage recovered is determined by the complexity of the library, the target protein, and the selection stringency. Accordingly, prior to the present invention three types of selection (shown in
FIG. 1
) have been evaluated over the last several years: 1) affinity selection against simple targets like immobilized proteins; 2) affinity selection against complex targets such as the cell surface; and 3) selection after phage processing such as their internalization by cells.
When utilizing affinity selection, unbound phage are washed away with buffers of different stringencies and the remaining attached phage particles are recovered, amplified in bacteria, and then further enriched by repeated rounds of adsorption and recovery. In early rounds of selection, specific binding phage may be present among millions, if not billions, of other phage particles depending on the complexity of the library. While the phage recovered may be present in extremely low concentrations, they must be recovered in an infective form in order to allow for amplification by infecting host bacteria. As the selection is repeated, the library is significantly reduced in complexity and phage encoding the binding ligands can then be characterized by DNA sequencing.
With the success of ligand selection using phage libraries screened against immobilized proteins, investigators next began to select against whole cells Hoogenboom et al.,
European J. of Biochem.
260:774-784, 1999; Szardenings et al.,
J. Biol. Chem.
272(44):27943-27948, 1997; Pereira et al.,
J. Immunol Meth
203(1):11-24, 1997; Pasqualini et al.,
Nature
380:364-366, 1996. A clear advantage of this kind of “biopanning” is that little or no prior knowledge of the target molecule (i.e. a receptor) is needed and it can be in its native form on the cell surface. But the fact that the target protein may be low in concentration relative to the other cell surface proteins presents a significant disadvantage to selection and as in affinity selection against immobilized targets, non-specifically adherent phage can give false positive signals. A low concentration of non-specific phage can interfere in the early rounds of selection when the true binders are in extremely low concentrations.
Despite these issues, the selection of peptides on complex targets has been successful. Recent studies by Pasqualini's laboratory, have extended this approach even further by demonstrating that organ homing peptides can be selected from libraries that are “biopanned” in-vivo. By applying standard phage display selection techniques to mice, in-vivo, they identified peptides capable of selectively targeting phage to the vasculature of different organs including, brain, prostate, and kidney. Pasqualini and Ruoslahti,
Mol. Psychiatry
1(6):423, 1996.
In an effort to increase selection stringency and overcome the problems of non-specific adsorption that are associated with biopanning against whole cells, alternative strategies have been explored. Hart et al. initially demonstrated that RGD targeted phage are internalized through receptor mediated endocytosis.
J. Biol. Chem.
269(17):12468-12474, 1994 Subsequently, Barry et al. showed that cell-specific internalizing peptides can be selected from large diverse libraries of displayed peptides by washing phage off the cell surface at low pH and recovering internalized phage from cell lysates.
Nat. Med.
2:299-305, 1996. However, these methods suffered from multiple steps as well as having no clear ability to determine in an initial screen which ligands would facilitate gene transduction and which would not. The original rationale behind selection by internalization was merely to increase the stringency of selection and therefore increase the ratio of signal to background.
Accordingly, current methodologies are inadequate to determine the usefulness of ligands for facilitating transfer and transduction of a cell by a nucleic acid molecule associated with the genetic package and ligand.
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 transfer.
Thus, current screening methods are inadequate for selecting peptide or protein ligands that bind to a cell surface receptor, internalize and lead to expression of the carried nucleic acid molecule. The present invention discloses display methods that select peptide or protein ligands that internalize and facilitate cell transduction and expression of product from an associated nucleic acid molecule, and further provides other related advantages.
SUMMARY OF THE INVENTION
The present invention utilized novel genetic package display of putative ligands to investigate the ability of these molecules to facilate cel
Baird Andrew
Kassner Paul
Larocca David
Ponnaluri Padmashri
Seed Intellectual Property Law Group PLLC
Selective Genetics Inc.
LandOfFree
METHODS USING GENETIC PACKAGE DISPLAY FOR DETECTING AND... 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 USING GENETIC PACKAGE DISPLAY FOR DETECTING AND..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and METHODS USING GENETIC PACKAGE DISPLAY FOR DETECTING AND... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3192754