Bacteriophage library displaying immunoglobulin repertoires with

Details Bacteriophage library displaying immunoglobulin repertoires with Bacteriophage library displaying immunoglobulin repertoires with

1997-09-04

2000-01-25

Railey, II, Johnny F.

Chemistry: molecular biology and microbiology

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

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

435 691, 435 697, 435 711, 4353201, 435472, 530350, 530402, 5303871, 5303873, C12N 701, C12N 1513, C07K 1646, C07K 1107

Patent

active

060177322

DESCRIPTION:

BRIEF SUMMARY
This is the U.S. National Phase of International Application No. PCT/GB84/01422, filed Jun. 30, 1994.
The present invention relates to members of specific binding pair (sbp) which comprise a binding site for complementary second sbp member. More particularly, it relates to sbp members, especially repertoires thereof, wherein a chemical moiety is incorporated specifically at an amino acid residue within the binding site. It also relates to the production of such repertoires and selection of such repertories and selection from a repertoire of first sbp member with binding specificity for a second sbp member of interest. The sbp member may be one wherein the binding site is formed by binding regions of immunoglobulin heavy and light chain variable domains.
It was disclosed in WO 92/01047 that polypeptides which are members of specific binding pairs, including antibody fragments, can be displayed on the surface of bacteriophage and that they will bind complementary members of the specific binding pair, e.g. antigen in the case of antibody. First sbp member with desirable binding properties-can be selected directly using this characteristic. The principles described therein have been subsequently extended in the further patent applications WO 92/20791; WO 93/06213; WO 93/11236 and WO 93/19172. Phage libraries have been prepared displaying antibody repertoires from for instance human peripheral blood lymphocytes and the spleens of immunized animals and specific antibodies isolated. In WO 92/01047, it is described how V gene repertoires for display on bacteriophage may be made in vitro by combining un-rearranged V genes, with D and J segments. Antibodies against a variety of antigens have been selected from such artificially rearranged V gene repertoires displayed on phage (H. R. Hoogenboom & G. Winter J. Mol. Biol. 227 381-388 1992; and as disclosed in WO 93/06213 and WO 93/11236).
However, the chemical diversity of these encoded libraries is restricted to natural amino acids. It would be desirable to be able to incorporate other reactive groups at the binding site of sbp members such as antibodies. These would have a number of applications.
The development of antibody based biosensors (reviewed by J. R. North, Trends Biotechnol. 3 180-186, 1985) has been limited by the ability to combine recognition and effector functions into the same molecule. Transduction of the antigen binding event would need to be mediated by alternative means such as fluorophores, luminophores or redox groups. These reporter groups should ideally be an integral part of the antibody binding site, such that antigen binding would lead to a change in the groups microenvironment, and a corresponding change in the fluorescence, luminescence or redox potential.
Similarly, the design of catalytic antibodies (reviewed by R. A. Lerner et al. Science 252 659-667, 1991) would be facilitated by the incorporation of nucleophilic groups and non-peptidyl cofactors such as metal ions, haems and flavins, a common feature of many enzymes, into the antibody binding site. These modifying groups would ideally be incorporated covalently at the antigen binding site, thus becoming integral parts of the paratope. Such chemically modified antibodies could then be used directly as detection agents in immunoassay applications, such as time resolved fluorescence immunoassay, or directly in catalysis.
In this application, we demonstrate that chemical groups can be covalently incorporated into a repertoire of sbp members displayed on phage and that sbp members can be selected from these repertoires with covalently bound groups at the antigen binding site. We have called such repertoires, chemisynthetic libraries.
The utility of the incorporation of chemical moieties at the antigen binding sites of antibody molecules has been recognized by previous workers, particularly for the derivation of catalytic antibodies. Rational protein engineering has been used (V. A. Roberts et al, Proc. Natl. Acad. Sci. 87 6654-6658 1990; B. L. Iversen et al Science 249 659-662, 1990; D. G.

REFERENCES:
Clackson et al., Nature 352:624-628 (1991).
Barbas III et al., Proc. Natl. Acad. Sci. USA 89:4457-4461 (1992).
Staunton et al., Protein Engineering 6(Suppl.):93 (1993).
Pollack et al., Science 242:1038-1040 (1988).

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