Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-06-04
2004-02-03
Celsa, Bennett (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S007100, C435S235100, C435S320100
Reexamination Certificate
active
06686154
ABSTRACT:
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
Phage display and related techniques have become powerful methods for the discovery of affinity binding reagents (Smith (1985)
Science
228: 1315-1317). Linear and constrained peptides, antibody fragments (e.g., scFvs, Fvs and Fabs), as well as a number of alternative binding domains have all been displayed on phage particles, for example, via fusion to one of the phage coat proteins. Although several phage proteins (derived from gVIII, gVI, gVII and gIX) have all been used as fusion partners for display of recombinant proteins, gIII is the most widely used. Phagemids containing a phage origin of replication, an antibiotic resistance marker, and a gene encoding a binding domain/gIII fusion protein are readily constructed via conventional molecular biology techniques. Through large-scale ligation and transformation as well as recombination strategies, large libraries of 10
8
to 10
11
different recombinants are now being generated for use in affinity selection strategies (de Haard et al. (1999)
J. Biol. Chem.
274: 18218-18230; Sblattero and Bradbury (2000)
Nat. Biotechnol
. 18: 75-80); Sheets et al. (1998)
Proc. Natl. Acad. Sci., U.S.A
. 95:6157-6162, published erratum appears in
Proc. Natl. Acad. Sci., U.S.A
. (1999) 96: 795).
Once a library of phage displaying potential binding agents is generated, individual phage with the capacity to bind to a chosen target must be isolated from an enormous excess of non-binding phage. To screen large numbers of phage to identify those that display polypeptides having a desired activity, it is desirable to develop high-throughput screening (HTS) methods. Preferably, such HTS methods would automate the phage screening process so that large numbers of phage could be screened with little human intervention. Although HTS methods are available for many types of screening, previously known phage display protocols include steps that are not readily automatable. In particular, phage display protocols require, prior to screening, separation of the phage from the host cells in which the phage are amplified.
Traditionally, overnight cultures of bacteria producing phage are centrifuged or filtered to pellet bacteria and phage supernatants are used in the screening (See generally, Kay et al., eds. (1996)
Phage display of peptides and proteins: a laboratory manual
. Academic Press Inc., San Diego Calif.). Alternatively, phage can be purified and concentrated from cleared supernatants by precipitation (e.g., with polyethylene glycol). However, these clearing methods are not readily performed by robotic systems (e.g., automated workstations). Therefore, time-consuming and expensive human intervention is required. These drawbacks are exacerbated as the numbers of samples are increased and during high-throughput screening. Therefore, a need exists for more fully automated methods for screening of phage display libraries. The present invention fulfills this and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods for screening a population of replicable genetic packages (e.g., phage, eukaryotic viruses, and the like) to obtain particles that display on their surface a fusion protein that specifically binds to a target molecule. Unlike previous methods, which involve clearing a culture of cells prior to screening the methods of the present invention involve contacting a target molecule with an uncleared cell culture that contains a population of replicable genetic packages. Each replicable genetic package displays on its surface a fusion protein that has a surface-displayed replicable genetic package polypeptide and a potential binding polypeptide. The replicable genetic package that specifically bind to the target molecule form complexes containing replicable genetic packages and target molecules. In some cases, the potential binding polypeptide can be encoded by a member of a library of nucleic acid molecules. For example, the nucleic acid molecules can be cDNA molecules or recombinant products. In other cases, the potential binding polypeptide can be, for example, an antibody, or derivative of an antibody. For example, the potential binding polypeptide can be a scFv or a Fab.
The methods of the invention are useful for obtaining polypeptides that bind to essentially any molecule. For example, the target molecule can be a polypeptide, an RNA, a DNA, a small organic molecule and a carbohydrate. The target molecules can be immobilized directly or indirectly to a solid support. Solid supports such as a bead, a chip, a microtiter plate, a eukaryotic cell, or a prokaryotic cell are present in some embodiments of the invention. The solid supports of the present invention can contain a variety of materials, such as Sepharose, polystyrene, glass, silicon oxide, etc.
In some embodiments, the methods also involve obtaining replicable genetic packages that specifically bind to the target molecule. For example, the replicable genetic packages that specifically bind to the target molecule can be separated from the bacterial cells after the binding of the phage to the target molecule. For example, the uncleared cell culture can be separated from a replicable genetic package-target complex(es) using aspiration. Once the replicable genetic packages are bound to the target molecule, some embodiments of the invention can further involve eluting the replicable genetic packages from the target molecule. Also, some embodiments involve identifying the replicable genetic packages that specifically bind to the target molecule with a detection reagent.
The present invention also provides compositions containing an uncleared cell culture, which contains: (a) a population of replicable genetic packages that display on their surfaces a fusion protein that includes a surface-displayed replicable genetic package polypeptide and a potential binding polypeptide; (b) a complex that is composed of one or members of the library of replicable genetic packages that specifically bind to the target molecule; and (c) cells in which the replicable genetic packages were amplified.
REFERENCES:
patent: 6451527 (2002-09-01), Larocca et al.
patent: WO 95/16027 (1995-06-01), None
patent: WO 95/34648 (1995-12-01), None
patent: WO 00/31246 (2000-06-01), None
Cabilly, S.; Heldman, J.; Katchalski-Katzir, E. “Screening Phage Display Peptide Libraries on Nitrocellulose Membranes” In: Combinatorial Peptide Library Protocols. Edited by S. Cabilly Totowa, New Jersey: Humana Press, 1998.*
Smith, G. P.; Scott, J. K. “Libraries of Peptides and Proteins Displayed on Filamentous Phage” In: Methods in Enzymology vol. 217. Edited by R. Wu. New York, Academic Press, Inc. 1993.*
Light, J.; Maki, R.; Assa-Munt, N. “Expression cloning of cDNA by phage display selection” Nucleic Acids Research, 1996, 24(21), 4367-4368.*
WO 99/57312 (Cahill et al) Nov. 11, 1999.*
Griffiths, et al. “Isolation of High Affinity Human Antibodies Directly From Large Synthetic Repertoires”Embo Journal(Jul. 1994) Vo. 13(14), pp. 3245-3260.
Haard, et al. “A Large Non-immunized Human Fab Fragment Phage Library That Permits Rapid Isolation and Kinetic Analysis of High Affity Antibodies”J. of Biological Chemistry(Jun. 1999) vol. 274(26), pp. 18218-18230.
Krebber, te al. “Selectivity-infective Phage (SIP): A Mechanistic Dissection of a Novel in vivio Selection for Protein-ligand Interactions”J. Molecular Biology(1997) vol. 268(3), pp. 607-618.
Phillps, Lisa M., “Detection of Antibody Display Phage Without Clearing of Bacterial Culture”Biotechniques(2000) vol. 26(4), pp. 737-740.
Sheets, et al., “Efficient Construction of a Large Nonimmune Phage Antibody Library: The Production of High-affinity Human Single-chain Antibodies to Protein Antibodies to Protein Antigen”Proc. Natl. Acad. Sci.(May 1998) vol. (95), pp. 6157-6162.
Kassner Paul D.
Nock Steffen
Celsa Bennett
Epperson Jon D.
Zyomyx, Inc.
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