Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1997-07-31
1999-11-23
Hutzell, Paula K.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
5303873, 5303871, 5303881, 435 691, C12P 2108, C12P 2106, C07K 1600, G01N 3353
Patent
active
059898305
DESCRIPTION:
BRIEF SUMMARY
This application is the national phase filing of International application PCT/EP96/03605, filed Aug. 14, 1996, which designated the U.S. and claims priority based on GB 95307332.7, filed in Great Britain on Oct. 16, 1995.
The invention relates to new bispecific or bivalent antibody fragment analogues, a process for preparing such antibody fragment analogues and various uses of such antibody fragment analogues.
BACKGROUND OF THE INVENTION AND PRIOR ART
1. Antibody Structure
Antibody molecules typically are Y-shaped molecules whose basic unit consist of four polypeptides, two identical heavy chains and two identical light chains, which are covalently linked together by disulfide bonds. Each of these chains is folded in discrete domains. The C-terminal regions of both heavy and light chains are conserved in sequence and are called the constant regions, also known as C-domains. The N-terminal regions, also known as V-domains, are variable in sequence and are responsible for the antibody specificity. The antibody specifically recognizes and binds to an antigen mainly through six short complementarity-determining regions located in their V-domains (see FIG. 1).
In this specification abbreviations are used having the following meaning. directly or via a peptide linker)
It is generally known that proteolytic digestion of an antibody with papain yields three fragments. The fragment containing the CH.sub.2 and CH.sub.3 domains of the two heavy chains connected by the complete hinge (see FIG. 1) crystallises very easily and was therefore called Fc fragment. The two other fragments are identical and were called Fab fragments, as they contained the antigen-binding site. Digestion with pepsin is such that the two Fab's remain connected via the hinge, forming only two fragments: Fc' and Fab.sub.2.
The Fv is the smallest unit of an antibody which still contains the complete binding site (see FIG. 1) and full antigen binding activity. It consists of only the V-domains of the heavy and light chains thus forming a small, heterodimeric variable fragment or Fv. Fv's have a molecular weight of about 25 kD, which is only one sixth of the parent whole antibody (in the case of an IgG). Previously Fv's were only available by proteolysis in a select number of cases (Givol, 1991). The production of Fv's can now be achieved more routinely using genetic engineering methods through cloning and expressing DNA encoding only the V-domains of the antibody of interest. Smaller fragments, such as individual V-domains (Domain Antibodies or dABs, Ward et al., 1989), and even individual CDR's (Williams et al., 1989; Taub et al., 1989) were shown to retain the binding characteristics of the parent antibody. However, this is not achievable on a routine basis: most naturally occurring antibodies need both a V.sub.H and a V.sub.L to retain full immunoreactivity. For example, in the case of V.sub.H D1.3 (Ward et al., 1989), although it still binds hen egg lysozyme (HEL) with an affinity close to that of the parent antibody, it was shown that loss of specificity was observed in that it can no longer distinguish turkey lysozyme from HEL, whereas the Fv can (Berry and Davies, 1992). Although murine dABs can be obtained more routinely from spleen libraries (Ward et al., 1989), the approach is unsustainable because of the many problems associated with their production and physical behaviour: expression is extremely poor, affinity tends to be low, stability and solubility in water is low, and non-specific binding is usually very high. According to the literature a possible explanation of these undesirable characteristics is the exposure of the hydrophobic residues which are normally buried in the V.sub.H -V.sub.L interface. The exposed hydrophobic patches are thought to contribute to aggregation of the protein inside the cells and/or in the culture medium, leading to poor expression and/or poor solubility (Anthony et al., 1992; Ward et al., 1989). The hydrophobic patches can also explain the high non-specific binding described by Berry and Davies, 1992
REFERENCES:
Burgess et al (J. Cell. Bio, III: 2129-2138), 1990.
Lazar et al (Mol & Cell Bio, 8: 1247-1252), 1988.
Tao et al (J. Immunol, 143: 2595-2601), 1989.
Bowie et al (Science 247: 1306-1310), 1990.
Klausner (Biotechnology, 4: 1042-1043), 1984.
Better et al (Science, 240: 1041-1043), 1988.
Holliger et al (PNAS, 30: 6444-6448), 1993.
Verhoeyen et al: "Antibody fragments for controlled delivery of therapeutic agents" Biochemical Society Transactions, vol. 23, No. 4, Jul. 18-21 1995, pp. 1067-1073 XP000565752, see the whole document.
Davis Paul James
van der Logt Cornelis Paul Erik
Verhoeyen Martine Elisa
Wilson Steve
Hutzell Paula K.
Ungar Susan
Unilever Patent Holdings BV
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