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...
Reexamination Certificate
1993-05-07
2001-03-20
Burke, Julie (Department: 1642)
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...
C530S387500, C530S387700, C424S133100, C424S137100, C424S138100, C435S069600, C435S069700, C435S091100, C435S328000, C435S329000, C435S330000, C536S023400, C536S023530
Reexamination Certificate
active
06204366
ABSTRACT:
This invention relates to specific binding agents, and in particular to polypeptides containing amino acid sequences that bind specifically to other proteinaceous or non-proteinaceous materials. The invention most particularly concerns the production of such specific binding agents by genetic engineering.
Antibody structure
Natural antibody molecules consist of two identical heavy-chain and two identical light-chain polypeptides, which are covalently linked by disulphide bonds.
FIG. 14
of the accompanying drawings diagramatically represents the typical structure of an antibody of the IgG class. Each of the chains is folded into several discrete domains. The N-terminal domains of all the chains are variable in sequence and therefore called the variable regions (V-regions). The V-regions of one heavy (VH) and one light chain (VL) associate to form the antigen-binding site. The module formed by the combined VH and VL domains is referred to as the Fv (variable fragment) of the antibody. The C-terminal ends of both heavy and light chains are more conserved in sequence and therefore referred to as the constant regions. Heavy chain constant regions are composed of several domains, eg. the heavy chain constant region of the gamma-isotype (IgG) consists of three domains (CH1, CH2, CH3) and a hinge region which connects the CH1 and CH2 domains. The hinges of the two heavy chains are covalently linked together by disulphide bridges. Light chains have one constant domain which packs against the CH1 domain. The constant regions of the antibody molecule are involved in effector functions such as complement lysis and clearing by Antibody Dependant Cell Cytotoxicity (ADCC). Classical digestion of an antibody with the protease papain yields three fragments. One fragment contains the CH2 and CH3 domains and, as it crystallises easily, was called the Fc fragment. The other two fragments were designated the Fab (antigen-binding) fragments, they are identical and contain the entire light chain combined with the VH and CH1 domain. When using pepsin, the proteolytic cleavage is such that the two Fabs remain connected via the hinge and form the (Fab)
2
fragment. Each of the domains is represented by a separate exon at the genetic level.
The variable regions themselves each contain 3 clusters of hypervariable residues, in a framework of more conserved sequences. These hypervariable regions interact with the antigen, and are called the Complementarity Determining Regions (CDRs). The more conserved sequences are called the Framework Regions (FRs). See Kabat et al (1987). X-ray studies of antibodies have shown that the CDRs form loops which protrude from the top of the molecule, whilst the FRs provide a structural beta-sheet framework.
Modified antibodies
In one embodiment, the invention relates to so-called “reshaped” or “altered” human antibodies, ie. immunoglobulins having-essentially human constant and framework regions but in which the complementarity determining regions (CDRs) correspond to those found in a non-human immunoglobulin, and also to corresponding reshaped antibody fragments.
The general principles by which such reshaped human antibodies and fragments may be produced are now well-known, and reference can be made to Jones et al (1986), Riechmann et al (1988), Verhoeyen et al (1988), and EP-A-239400 (Winter). A comprehensive list of relevant literature references is provided later in this specification.
Reshaped human antibodies and fragments have particular utility in the in-vivo diagnosis and treatment of human ailments because the essentially human proteins are less likely to induce undesirable adverse reactions when they are administered to a human patient, and the desired specificity conferred by the CDRs can be raised in a host animal, such as a mouse, from which antibodies of selected specificity can be obtained more readily. The variable region genes can be cloned from the non-human antibody, and the CDRs grafted into a human variable-region framework by genetic engineering techniques to provide the reshaped human antibody or fragment. To achieve this desirable result, it is necessary to identify and sequence at least the CDRs in the selected non-human antibody, and preferably the whole non-human variable region sequence, to allow identification of potentially important CDR-framework interactions.
Antibodies raised against the human milk fat globule (HMFG), generally in a delipidated state, can exhibit a broad spectrum of reactivity with epithelial origin neoplasms, particularly carcinomas of the breast, ovary, uterus and lung. See Taylor-Papadimitriou et al (1981) and Arklie et al (1981). One well-characterised antibody (designated HMFG1) is known to bind to a component of the HMFG, also found in some body tissues, some cancer tissues and urine, which has been designated polymorphic epithelial mucin (PEM) (Gendler et al, 1988). Binding is thought to involve the peptide core of the PEM. Corresponding useful specificity can be achieved by raising antibodies against cancer cells, for example breast cancer cell lines.
EP-A2-0369816 (The University of Melbourne, Xing et al) describes monoclonal antibodies specific for human polymorphic epithelial mucin, which bind to a defined amino acid sequence. It is suggested in EP-A2-0369816 that the described antibodies may be “humanised” according to the method of Riechmann et al (1988). However, Xing et al do not describe the actual preparation of any such reshaped anti-PEM antibodies.
SUMMARY OF THE INVENTION
The invention provides, as one embodiment, a synthetic specific binding polypeptide having specificity for a polymorphic epithelial mucin (PEM), and especially a synthetic specific binding polypeptide having anti-human milk fat globule (HMFG) specificity, containing one or more of the CDRs depicted in
FIGS. 1 and 2
of the accompanying drawings. By synthetic, we particularly mean that the polypeptide is produced by recombinant DNA technology, and to that extent at least is different from a naturally-occurring or naturally-induced specific binding agent having identical specificity. Alternatively, the synthetic polypeptide has been produced by artificially assembling a sequence of amino acids to produce a novel or nature-identical molecule. The synthetic polypeptide can be equivalent to an intact conventional antibody, or equivalent to a multiple or. single-chain fragment of such an antibody, or can be simply a material that includes one or more sequences that confer the desired specific binding capability.
The invention provides as an important embodiment a reshaped human antibody, or a reshaped human antibody fragment, having anti-PEM specificity, and especially having anti-HMFG specificity, containing one or more of the CDRs depicted in
FIGS. 1 and 2
of the accompanying drawings. Preferably, the reshaped antibody or fragment of the invention contains all 3 of the CDRs depicted in
FIG. 1
of the accompanying drawings, in a human heavy chain variable region framework. Alternatively, or in addition, the reshaped antibody or fragment of the invention contains all 3 of the CDRs depicted in
FIG. 2
of the accompanying drawings, in a human light chain variable region framework.
Another embodiment of the invention is a reshaped antibody or reshaped antibody fragment containing a protein sequence as depicted in FIG.
12
and/or
FIG. 13
of the accompanying drawings.
Other important embodiments of the invention are an expression vector incorporating a DNA sequence as depicted in FIG.
12
and/or
FIG. 13
of the accompanying drawings, and an expression vector incorporating a DNA sequence encoding one or more of the protein sequences designated as being a CDR in FIG.
1
and/or
FIG. 2
of the accompanying drawings.
An important aspect of the invention is a stable host cell line containing a foreign gene that causes the host cell line to produce a specific binding agent according to the invention. This can be a stable host cell line containing a foreign gene that encodes at least one of the amino acid sequences designated as being a CDR
Burke Julie
Pillsbury Madison & Sutro LLP
Unilever Patent Holdings B.V.
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