Recombinant antibodies at the surface of E. coli

Details Recombinant antibodies at the surface of E. coli Recombinant antibodies at the surface of E. coli

1993-05-10

1997-01-07

Feisee, Lila

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

435 696, 4353201, 4352523, 435 712, C12P 2108, C12P 2104, C12N 512, C12N 120

Patent

active

055916040

DESCRIPTION:

BRIEF SUMMARY
The invention concerns vectors that express single chain antibodies coupled to the surface of antibody-producing cells. Furthermore, the invention embraces the use of these vectors to rapidly isolate single cells producing specific antibodies. Finally, the invention provides a method for the isolation of cells producing specific antibodies.
Previous screening procedures for recombinant antibodies have employed ELISA assays of bacterial supernatants (Nature 341, 544-546 (1989)) or radioactively labeled immunogens for screening nitrocellulose plaque lift-offs of bacterial colonies infected with phage expression vectors (Science 246, 1275-1281 (1989); Proc. Natl. Acad. Sci. USA 87, 6450-6454 (1990); Proc. Natl. Acad. Sci. USA 87 8095-8099 (1990)). However, for the selection of specific antibodies from randomly combined light and heavy chain libraries that do not contain a preponderance of antibodies to a particular antigen, the task of screening millions of antibody clones would be greatly facilitated by targeting antibodies to the surface of bacteria or viruses. Immobilized antigens could then be used to select specific antibodies.
A viral system for the surface presentation of antibodies has recently been disclosed in Nature, 348, 552-554 (1990). Single chain variable domains (the precursors of applicant's own antibody construct) were fused to the docking protein (protein p III) of phage particles. Phages bearing the fusion protein were able to be enriched on columns of antigen.
However, these fusion phages have been shown to be mainly useful for displaying relatively small inserts, probably because the larger inserts have an adverse effect on the infectivity function of p III. Libraries of "phage antibodies", therefore, run the risk of being quickly dominated by deletion mutants. Furthermore, relatively large numbers of phage particles appear to bind unspecifically to columns of immobilized antigen.
Thus the technical problem underlying this invention is to provide a more efficient mean for screening antibody libraries in bacteria.
This problem is solved by providing a vector that expresses single chain antibody variable domains coupled to the peptidoglycan associated lipoprotein of E. coli (PAL, J. Biochem. 86, 991-1000 (1979); Eur. J. Biochem., 163, 73-77 (1987)).
PAL is a cell envelope component of E. coli that is particularly resistant to solubilization by SDS (J. Biochem. 86, 991-1000 (1979)). Its peptidoglycan associated protein component has a molecular mass of 16600 (Eur. J. Biochem., 163, 73-77 (1987)) and is modified at the amino-terminal cysteine by a lipid moiety that is integrated into the outer membrane. The attachment of antibodies to the amino-terminus of PAL therefore is a means of presenting them for antigen binding at the cell surface.
The antibody-PAL fusion protein was identified on the cell surface by a monoclonal antibody to an epitope in the linker sequence between the heavy and light chains. It was able to bind antigen and was tightly bound to the murein layer of the cell envelope. Immunofluorescence studies on unfixed cells showed that functional antibody domains were quite accessible at the cell surface.
A major attraction of the bacterial system of this invention is that an abundance of antibody molecules are available for binding in contrast to the five antibody fusion molecules that are bound per particle in the phage system. The use of immunofluorescently labeled antigens should therefore provide a rapid means of isolating cells producing specific antibodies. Screening with a fluorescence assisted cell sorter (FACS), for example, could theoretically provide an enrichment of at least 10.sup.6 in one step since more than several million bacteria can be scanned at one time and single bacteria can be selected. Alternatively, bacteria expressing specific antibodies could be selected by binding to immobilized antigens. Further advantages include the efficiency of transformation, the ease of propagation and the absence of selection pressure to remove antibody DNA during library ampl

REFERENCES:
Nature 341, 544-546 (1989).
Science 246, 1275-1281 (1989).
Proc. Natl. Acad. Sci. USA 87, 6450-6454 (1990).
Proc. Natl. Acad. Sci. USA 87, 8095-8099 (1990).
Nature 348, 552-554 (1990).
J. Biochem 86, 991-1000 (1979).
Eur. J. Biochem. 163, 73-77 (1987).
Nature 332, 323-327 (1988).
J. Mol. Biol. 189, 367-370 (1986).
J. Bacteriol. 169, 4379-4383 (1987).
Science 240, 1040-1043 (1988).
J. Biochem. 86, 979-989 (1979).
Science 233, 747-753 (1986).
Proteins: Structure, Function and Genetics, vol. 8, No. 4 1990, Wiley Pre NY, US; pp. 309-314.
Nature 349, 293-299 (1991).
Biotechnology vol. 9, No. 12 (1991), pp. 1369-1372 Fuchs et. al.

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