Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1995-05-30
1998-12-01
Scheiner, Toni R.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
536 2353, 435328, C12P 2108, C07K 1600
Patent
active
058440941
DESCRIPTION:
BRIEF SUMMARY
This invention relates to the construction, application and production of novel polypeptides with enhanced or modified binding activity or specificity to haptens and antigens.
The invention also relates to the construction, modification and selection of recombinant antibody-like molecules derived from expression of libraries of surface presenting antigen- or hapten-binding moieties, and to uses of these molecules.
The polypeptides of the invention have utility in the diagnostic, therapeutic, predictive or preventative fields of the pharmaceutical and health care industries, as well as more general application in the detection and assay of chemical entities.
BACKGROUND OF THE INVENTION
Antibodies are protein molecules which possess a binding affinity for a target antigen or hapten. Due to the specificity of the binding interaction, antibodies are commonly used as diagnostic and therapeutic reagents. Monoclonal antibodies are derived from a pure cell line such as hybridoma cells; however, the hybridoma technology is expensive, time-consuming to maintain and limited in scope. It is not possible to produce monoclonal antibodies, much less antibodies of the appropriate affinity, to a complete range of antigens.
Antibody genes or fragments thereof can be cloned and expressed in E. coli in a biologically functional form. Antibodies and antibody fragments can also be produced by recombinant DNA technology using either bacterial or mammalian cells. In the Fab region of an antibody, the combination of the two heavy and light chains provides six variable surface loops at the extremity of the molecule. These loops in the outer domain (Fv) are termed complementarity-determining-regions (CDRs), and provide the specificity of binding of the antibody to its antigenic target. This binding function is localised to the variable domains of the antibody molecule, which are located at the amino-terminal end of both the heavy and light chains. This is illustrated in FIG. 1. The variable regions of some antibodies remain non-covalently associated (as V.sub.H V.sub.L dimers, termed Fv regions) even after proteolytic cleavage from the native antibody molecule, and retain much of their antigen recognition and binding capabilities. Methods of manufacture of two-chain Fv substantially free of constant region are disclosed in U.S. Pat. No. 4,642,334
Recombinant Fv fragments are prone to dissociation, and therefore some workers have chosen to covalently link the two domains to form a construct designated scFv, in which two peptides with binding domains (usually antibody heavy and light variable regions) are joined by a linker peptide connecting the C-terminus of one domain to the N-terminus of the other, so that the relative positions of the antigen binding domains are consistent with those found in the original antibody (see FIG. 1).
Methods of manufacture of covalently linked Fv fragments are disclosed in U.S. Pat. No. 4,946,778 and U.S. Pat. No. 5,132,405. Further heterogeneity can be achieved by the production of bifunctional and multifunctional agents (Huston et al U.S. Pat. No. 5,091,513, and Ladner et al U.S. Pat. No. 4,816,397).
The construction of scFv libraries is disclosed for example in European Patent Application No. 239400 and U.S. Pat. No. 4,946,778. However, single-chain Fv libraries are limited in size because of problems inherent in the cloning of a single DNA molecule encoding the scFv. Non-scFv libraries, such as V.sub.H or Fab libraries, are also known, (Ladner and Guterman WO 90/02809), and may be used with a phage system for surface expression (Ladner et al WO 88/06630 and Bonnert et al at WO 92/01047).
For use in antibody therapy, monoclonal antibodies, which are usually of mouse origin, have limited use unless they are first "humanised", because they elicit an antigenic response on administration to humans. The variable domains of an antibody consist of a .beta.-sheet framework with six hypervariable regions (CDRs) which fashion the antigen-binding site. Humanisation consists of substituting mouse sequences t
REFERENCES:
Bird et al., Single chain antiody variable regions, TIBTECH, vol. 9, Apr., 1991, pp. 132-137.
Bird et al., Single-Chain Antigen-Binding Proteins, Science, vol. 242 Oct. 21, 1988, pp. 423-426.
Huston et al., Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue producted in Escherichia coli. Proc. Natl. Acad. Sci. USA vol. 85, pp. 5879-5883.
Colcher et al. In vivo Tumor Targeting of a Recombinant Single-Chain Antigen-Binding Protein, Journal of the National Cancer Institute, vol. 82, No. 14, Jul. 18, 1990.
Sharon, "Structural correlates of high antibody affinity: Three engineered amino acid substitutions . . . ", Proc. Natl. Acad. Sci. USA 87: 4814-4817 (1990).
Lehninger, "Principles of Biochemistry", Worth Publishers, Inc., New York, 1982, Chapter 30, pp. 926-933.
Erlich, "PCR Technology, Principles and Applications for DNA Amplification", Stockton Press, New York, 1989, Part 1, pp. 1-5.
Callow, "Measurement of Antibodies to Influenza Virus Neuraminidase by an Enzyme-Linked Immunosorbent Assay", Infection and Immunity, vol. 41(2) Aug. 1983, pp. 650-656.
Sorvillo et al., "Preparation and Characterization of Monoclonal Antibodies Specific for Human Transforming Growth Factor Alpha", Chemical Abstracts, vol. 113, No. 1, 1990, abstract No. 4322f.
Callow, K.A. 1983 Infection & Immunity, 41:650-656.
Schaaper, R.M. 1988 PMAS, USA 85:8126-8130.
Pack, P. et al. Biochemistry 31(6):4570-4584, 1992.
McCartney et al. J. Protein Chemistry 10(6):669-683, 1991.
George, AJT et al. J. Cell Biochem. Suppl. 15 (Part E), p. 127, 1991.
Whitlow, H. et al. Methods 2(2):97-105, 1991.
Atwell John Leslie
Colman Peter Malcolm
Hudson Peter John
Irving Robert Alexander
Kortt Alex Andrew
Commonwealth Scientific and Industrial Research Organization
Scheiner Toni R.
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