Phage antibodies

Details Phage antibodies Phage antibodies

1998-05-26

2001-07-24

Bansal, Geetha P. (Department: 1642)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S006120, C435S069600, C435S069900, C435S320100, C435S332000, C436S546000, C436S547000, C530S387100, C530S395000

Reexamination Certificate

active

06265150

ABSTRACT:

BACKGROUND OF THE INVENTION
The construction of libraries of fragments of antibody molecules that are expressed on the surface of filamentous bacteriophage and the selection of phage antibodies (Phabs) by binding to antigens have been recognized as powerful means of generating new tools for research and clinical applications. This technology, however, has been mainly used to generate Phabs specific for purified antigens that are available in sufficient quantities of solid-phase dependent selection procedures. The effectiveness of such Phabs in biochemical and functional assays varies; typically, the procedure used to select Phabs determines their utility.
Typically, many antigens of interest are not available in pure form in very large quantities. This clearly limits the utility of Phabs in binding such materials for research and clinical applications. Further, the utility of Phabs in such applications is directly proportional to the purity of the antigens and purification methods to assure the specificity of the isolate Phabs. Human monoclonal antibodies that bind to native cell surface structures are expected to have broad application in therapeutic and diagnostic procedures. An important extension of phage antibody display technology would be a strategy for the direct selection of specific antibodies against antigens expressed on the surface of subpopulations of cells present in a heterogenous mixture. Ideally, such antibodies would be derived from a single highly-diverse library containing virtually every conceivable antibody specificity.
SUMMARY OF INVENTION
A library was constructed from 49 human germline V
H
genes fused to a J
H
4 gene and partly randomized CDR3 regions varying in length between 6 and 15 amino acids. The CDR3 regions were designated to contain short stretches of fully randomized amino acid residues flanked by regions of limited variability. Residues in the latter portion of CDR3 were selected based on their frequent occurrence in CDRs (complementarity-determining regions) of natural antibody molecules, random CDR3 with an increased frequency of clones producing functional antigen binding sites. The synthetic V
H
segments were combined with seven different V
L
genes and expressed as geneIII-scFv fragments on the surface of phage, resulting in a library of 3.6×10
8
clones. This library was used to isolate monoclonal phage antibodies (MoPhabs) to a variety of different structures (haptens, proteins and polysaccharides) by selection on solid phase-bound antigen.
Further, MoPhabs were also isolated by flow cytometry, resulting in MoPhabs specific for subpopulations of cells present in a heterogenous mixture. These antibodies detect known and novel structures on various populations of blood and fetal bone marrow cells.
DETAILED DESCRIPTION OF INVENTION
The phage antibodies of the instant invention are obtained from a library of phage antibodies which possess specificity for a plurality of antigens. In practice, such libraries can be obtained from a variety of sources or constructed by known methods. A method particularly useful for constructing such libraries is described in paper by G. Winter, et al.,
Annual Reviews of Immunology,
12, 433-455 (1994), which is incorporated by references.
The library is then admixed with the antigens (as used herein, antigen shall be inclusive of haptens and antigen analogs) of interest and the phage antibodies bound to these antigens are then isolated. The procedure may be repeated until a population of phage antibodies having the desired specificity(ies) is obtained, and the isolated phage antibodies may then be cloned by conventional methods known to those in the art.
In a preferred embodiment, the phage antibody library is admixed with a cell mixture labeled with a fluorescent labeled antigen, or a plurality of antigens each labeled with a different fluorescent label, and sorted by flow cytometry. Preferred labels include phycoerythrin (PE), PerCP, and fluorescein isothiocyanate (FITC). The phage antibodies bound to cells, thus obtained, can be eluted. The phage antibodies (phages that express antibody specificities of interest) can then be cloned by conventional techniques to obtain a plurality of phage antibodies having high specificity for single antigens.


REFERENCES:
patent: 0 610 774 A1 (1994-08-01), None
patent: WO 93/1123 A1 (1993-06-01), None
patent: WO 94/26787 (1994-11-01), None
patent: 9426787 (1994-11-01), None
deKruif et al PNAS 92 pages 3938-3942, 1995 Apr.*
de Kruif et al., “Leucine Zipper Dimerized Bivalent and Bispecific scFv Antibodies from a Semi-synthetic Antibody Phage Display Library,” J. of Biological Chemistry, 271:7630-7634 (1996).
de Kruif et al., “Rapid Selection of Cell Subpopulation-Specific Human Monoclonal Antibodies from a Synthetic Phage Antibody Library,” Proc. Natl. Acad. Sci. USA, 92:3938-3942 (1995).
de Kruif et al., “Selection and Application of Human Single Chain Fv Antibody Fragments from a Semi-synthetic Phage Antibody Display Library with Designed CDR3 Regions,” J. Mol. Biol. 248:97-105 (1995).
de Kruif et al., “New Perspectives on Recombinant Human Antibodies,” Immunology Today, 17:453-455 (1996).

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