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
1994-07-22
1999-06-08
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...
4241341, 4241851, 4241921, 4241301, 435328, 436547, 530328, C12P 2108, C07K 1646
Patent
active
059105739
DESCRIPTION:
BRIEF SUMMARY
The present invention describes a new class of antigen binding molecules which contain Fv-fragments of an antibody but do not use the constant antibody domains. They can also dimerize with other antibody fragment molecules or with ace non-antibody fragment molecules to form bi- or multifunctional antibody-fragment fusion proteins and so-called mininantibodies, respectively. The new fusion proteins can be used in the broad field of diagnostic and therapeutical medicine.
BACKGROUND OF THE INVENTION
Since a few years there is a great interest in the biotechnological field to modify naturally occurring antibodies in order to obtain more specified and more individual antibody species. Therefore, attempts have been made to produce (modified) antibody fragments.
All naturally occurring antibodies of all classes have at least two binding sites. This enables them to bind to a surface with a greater functional affinity (also called avidity) than monovalent fragments, such as Fab fragments. Over the last few years, methods have been described (Skerra and Pluckthun, 1988, Science 240, 1038-1040; Better et al., 1988, Science 240, 1041-1043) with which functional antibody fragments can be produced in Escherichia coli. These include the Fv fragment (the heterodimer consisting of V.sub.H and V.sub.L) and the Fab fragment (consisting of the complete light chain with the domains V.sub.L and C.sub.L as well as the first two domains of the heavy chains V.sub.H and C.sub.H1).
The Fv fragment, however, has a tendency to dissociate into V.sub.H and V.sub.L and therefore, it is advantageous to link the two domains covalently. One particular way of linking them is by designing a peptide linker between them, either in the orientation V.sub.H -linker-V.sub.L or V.sub.L -linker-V.sub.H (Bird et al.,1988, Science 242, 423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85, 5879) The resulting fragments are called single-chain Fv fragments.
All these fragments are, however, monovalent. We describe in this invention a method to engineer small dimerization domains based on peptides forming amphipathic helices. These peptides will be referred to as "intercalating", but this term is only meant to express the ability of targeted association and not a restriction referring to a particular structure of the dimerization interface.
While the methodology described here, is in principle applicable to either Fab, Fv or scFv fragments, it is the latter for which their use is most advantageous. In this case bivalent fragments can be constructed of very small size, and still the dissociation into V.sub.L and V.sub.H as well as the wrong matching of fragment chains, e.g. V.sub.L --V.sub.L, can be prevented.
Antibody fragments of small size are of particular advantage in many applications. In diagnostic applications (e.g. ELISA, RIA, etc.), the smaller molecules surface decreases the problems of nonspecific interactions, which are known to frequently involve the constant domains. The same is true in using antibody fragments as ligands in affinity chromatography. In tumor diagnostics or therapy, it is important that a significant proportion of the injected antibody penetrates tissues and localizes to the tumor, and is dependent on the molecular dimensions (Colcher et al., 1990, J. Natl. Cancer Inst. 82, 1191-1197). Expression yields and secretion efficiency of recombinant proteins are also a function of chain size (Skerra & Pluckthun, 1991, Protein Eng. 4, 971) and smaller proteins are preferred for this reason. Therefore, molecules of a small size are advantageous for several reasons.
Previously, decreasing the molecular dimensions of the antibody meant the preparation of proteolytic fragments. The smallest bivalent fragments, (Fab)'.sub.2 fragments, are still about twice the size of the present fragments of this invention. Therefore, these new fragments combine three features: (a) small size, (b) bivalence or bifunctionality and (c) ability of functional expression in E. coli.
There is great interest in bifunctional antibodies in a large number of
REFERENCES:
patent: 5132405 (1992-07-01), Huston et al.
Bird et al., "Single Chain Antibody Variable Regions," Trends in Biotech., vol. 9, pp. 132-137 (Apr. 1991).
Blondel et al., "Engineering the Quaternary Structure of an Exported Protein with a Leucine Zipper," Protein Engineering, vol. 4, No. 4, pp. 457-461 (1991).
Pack et al., "Miniantibodies: Use of Amphipathic Helices to Produce Functional, Flexibly Linked Dimeric F.sub.v Fragments with High Avidity in Escherichia coli," Biochemistry, vol. 31, No. 6, pp. 1579-1584 (Feb. 18, 1992).
Pack Peter
Pluckthun Andreas
Merck Patent Gesellschaft mit beschrankter Haftung
Scheiner Toni R.
Worrall Timothy A.
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