Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-02
2002-07-16
Ponnaluri, Padmashri (Department: 1643)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S069100, C435S320100, C435S091500, C435S091500, C435S091500, C435S091500, C435S091500, C435S091500, C536S023100, C536S023530
Reexamination Certificate
active
06420113
ABSTRACT:
BACKGROUND
Over recent years, many publications have reported the use of phage-display technology to produce and screen libraries of polypeptides for binding to a selected target. See, e.g, Cwirla, et al.,
Proc. Natl. Acad. Sci. USA
87:6378-6382 (1990); Devlin, et al.,
Science
249:404-406 (1990), Scott & Smith,
Science
249:386-388 (1990); Ladner, et al., U.S. Pat. No. 5,571,698. A basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide. The establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target bind to the target and these phage are enriched by affinity screening to the target. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means.
Phage display technology has also been used to produce and screen libraries of heterodimeric proteins, such as Fab fragments. See e.g., Garrard, et al.,
Bio/Tech
9:1373-1377 (1991). Phage display libraries of Fab fragments are produced by expressing one of the component chains as a fusion with a coat protein, as for display of single-chain polypeptides. The partner antibody chain is expressed in the same cell from the same or a different replicon as the first chain, and assembly occurs within the cell. Thus, a phage-Fab fragment has one antibody chain fused to a phage coat protein so that it is displayed from the outer surface of the phage and the other antibody chain is complexed with the first chain.
In a further expansion of the basic approach, polypeptide libraries have been displayed from replicable genetic packages other than phage. These replicable genetic packages include eukaryotic viruses and bacteria. The principles and strategy are closely analogous to those employed for phage, namely, that nucleic acids encoding antibody chains or other polypeptides to be displayed are inserted into the genome of the package to create a fusion protein between the polypeptides to be screened and an endogenous protein that is exposed on the cell or viral surface. Expression of the fusion protein and transport to the cell surface result in display of polypeptides from the cell or viral surface.
Although conventional display methods have achieved considerable success in isolating antibodies and other polypeptides with specific binding to selected targets, some inefficiencies and limitations remain. In conventional methods, many library members bind nonspecifically to the target or the solid phase bearing the target and are amplified along with specifically bound library members causing poor efficiency at each round of affinity selection. Not only can this waste time and effort in performing many rounds of affinity selection, but members bearing polypeptides having specific affinity are lost at each round. Selection is generally terminated when sufficient rounds of affinity selection have been performed to achieve a significant number of members bearing polypeptides with affinity for a target even though many nonspecifically binding members are still present. Clonal isolates are then picked and tested individually to reduce the risk of losing specific-binding members through further rounds of selection. Clonal isolates shown to bind specifically may then be cloned into an expression work for future analysis, and, large-scale production. Accordingly, only one or a few of library members bearing polypeptides with specific affinity for the target present in the original repertoire are ever isolated.
The present application provides inter alia novel methods that overcome these inefficiencies and difficulties, and produce new diagnostic and therapeutic reagents.
SUMMARY OF THE INVENTION
The invention provides libraries of at least four chimeric antibodies. At least 50% of the antibodies in the library have specific affinity for the same target and no library member constitutes more than 25% of the library. In some libraries, the antibodies are Fab fragments. In some libraries, the antibodies are single-chain antibodies. In some libraries, the antibodies are intact antibodies. Typically, a chimeric antibody light chain comprises a nonhuman light chain variable region and a human light chain constant region. Typically, a chimeric antibody heavy chain comprises a nonhuman heavy chain variable region and a human heavy chain constant region. In some libraries at least some antibodies have light and heavy chain variable regions that are randomly associated.
The invention further provides libraries of at least four different nucleic segments encoding chimeric antibody chains comprising a variable region and a constant region from different species. At least 90% of segments in the library encode chimeric antibody chains showing specific affinity for the same target and no library member constitutes more than 50% of the library. Some such libraries comprise at least four pairs of the different nucleic acid segments, the members of a pair respectively encoding heavy and light chimeric antibody chains, wherein at least 90% of the pairs encode heavy and light chimeric antibody chains that form complexes showing specific affinity for the same target, and no pair of nucleic acid segments constitutes more than 50% of the library. In some libraries, at least 95% of library members encode chimeric antibody chains forming complexes having specific affinity for the target and no member constitutes more than 25% of the library. Some libraries have at least 100 different members. In some libraries, each pair of nucleic acids is expressed as a discistronic transcript.
The invention further provides methods of producing a polypeptide library having affinity for a target. Such methods entail providing a library of replicable genetic packages, wherein a member comprises a replicable genetic package capable of displaying an antibody chain encoded by a genome of the package and the antibody chain varying between members. One then subclones a mixed population of DNA molecules encoding at least four different antibody chains of the library of replicable genetic packages into multiple copies of an expression vector to produce modified forms of the expression vector. One then introduces the modified forms of the expression vector into a host and expressing the antibody chains as chimeric antibody chains in the host, wherein a library of at least four different chimeric antibody chains are expressed, at least 90% of modified forms of the expression vector encode chimeric antibody chains having specific affinity for a target and no modified form of the expression vector constitutes more than 50% of the total forms.
The invention further provides methods of producing an antibody library having affinity for a target as follows. A library of phage is provided in which a member of the library comprises a phage capable of displaying from its outersurface an antibody comprising an antibody heavy chain variable domain complexed with an antibody light chain variable domain, in which either the heavy or light chain variable domain is expressed as a fusion protein with a coat protein of the phage and the heavy and light chain variable domains are encoded by the genome of the phage, and the heavy and light chain varies between members. A mixture of DNA molecules encoding the heavy and light chain variable domains from the phage library members are subcloned into an expression vector to produce modified forms of the expression vector. The modified forms of the expression vector are then into a host and expressed to produce the heavy and light chain variab
Buechler Joe
Gray Jeff
Valkirs Gunars
Biosite Diagnostics, Inc.
Ponnaluri Padmashri
Townsend and Townsend / and Crew LLP
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