Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Structurally-modified antibody – immunoglobulin – or fragment...
Reexamination Certificate
1996-03-22
2003-07-08
Saunders, David (Department: 1644)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Structurally-modified antibody, immunoglobulin, or fragment...
C530S387300
Reexamination Certificate
active
06589527
ABSTRACT:
The present invention relates to retargetting of antibodies to a site or antigen for which they have no functional specificity under normal circumstances. A method is described employing an antigen-specific binding substance which possesses at least two specificities; one specificity for the target site, the other capable of binding to part of an antibody molecule. In this manner, antibodies with no specificity for the antigen target may be brought into proximity with the antigen via the antigen-specific binding substance. This principle is advantageous for re-targeting antibodies in the circulation to sites of disease within the body, e.g. tumours or sites of is viral, bacterial or parasitic infection or combinations thereof. This principle may also be applied to block inappropriate immune responses exemplified by autoimmune disease or hypersensitivity reactions. Retargetting can be achieved with conventional bispecific antibodies, e.g. prepared chemically or from hybrid hybridomas, or using the novel bispecific antibody fragments, diabodies (P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993 and PCT/GB93/02492).
Antibodies are proteins elaborated by B-lymphocytes to play a key role in the specific arm of the vertebrate immune system. This arises from their collective capacity to bind to an enormous diversity of antigen structures, with individual antibody molecules capable of precise specificity for their cognate antigen. The bulk of the antibody population is found in abundance in the blood and interstitial fluids, with minor types located at mucosal surfaces such as the intestinal lumen. An antibody binding to a foreign organism or a tumor cell marks it for destruction by the antibody encoded effector functions of the immune system. Destruction may be effected by either the complement cascase or antibody directed cell-mediated cytotoxicity (ADCC). ADCC is mediated through binding of antibody Fc regions to their Pc receptors on e.g. macrophages, eosinophils, K cells but also basophils and mast cells. Interaction with Fc receptors mediates not only cytolysis but also phagocytosis and immune clearance. Ig isotypes differ markedly in the spectrum of effector functions they recruit.
The immune system operates natural checks and balances to prevent production of antibodies with specificity for the host, so-called ‘self-antigens’. occasionally, the system breaks down causing autoimmune disease. Self-tolerance is one reason why the immune system may not destroy tumours and other malignancies, since these derive from host cells growing abnormally.
It has proved possible to use antibodies in medical intervention, using antibodies manufactured outside the body. Techniques for immortalisation of B-lymphocytes has enabled manufacture of monoclonal antibodies for a range of commercial applications in science and human health-care (Clinical Applications of Monoclonal Antibodies, E. S. Lennox, Ed. British Medical Bulletin 1984. Churchill-Livingstone). Moreover, an understanding of the genetic and physical structure of antibodies has enabled their manipulation outside of the immune system, through the use of molecular biology techniques, especially using phage display technology (WO 92/01047; WO 92/20791; WO 93/06213; WO 93/11236; WO 93/19172; WO 94/13804).
Structurally, the simplest antibody (IgG) comprises four polypeptide chains inter-connected by disulphide bonds. The light chains exist in two different forms called kappa (K) and lambda (X). Each chain has a constant region (C) and a variable region (V). Each chain is organised into a series of domains. The light chains have two domains, one corresponding to the C-region (CL) and the other to the V-region (VL). The heavy chains have four domains, one V-region domain (VH) and three C-region domains, CH1, CH2 & CH3. The basic IgG antibody is Y-shaped; the two arms (tip of the Y, each being an ‘Fab’ region) contain a VH and a VL domain associated with one another. It is this pair of V-regions that differ from one antibody to another (owing to amino acid sequence variations), and which together are responsible for recognising the antigen and providing an antigen binding site (ABS). In even more detail, each V-region (whether heavy chain or light chain) consists of three complementarity determining regions (CDRs) separated by four framework regions (FR) The CDR's are the most variable part of the variable regions, and they perform the critical binding function. The CDR regions are derived from many potential germline sequences via a complex process involving recombination, mutation and selection.
It has been shown that the function of binding antigens can be performed by fragments of a whole antibody. Example binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) bispecific single chain Fv dimers (PCT/US92/09965) and (viii) diabodies, bivalent or bispecific fragments constructed by gene fusion (P. Holliger et al, supra; WO 94/13804. Diabodies are discussed further infra. Bispecific fragments are especially well suited to the current invention.
Whereas the V-domains (and fragments containing V-domains) are largely responsible for interacting with antigen, the C-domains recruit effector functions. The type of effector function recruited is largely governed by the class of C-domain (the isotype; M. Bruggemann et al J. Exp. Med. 166 1351 1987; L. Riechmann et al Nature 332 323 1988; J. Greenwood et al Eur. J. Immunol. 23 1098-1104 1993). In this way, antibodies, which have evolved to combat pathogens, bind to antigens on the pathogen and in so doing initiate an appropriate immune response aimed at destroying the invader. For example, C-domains of the IgG1 (&ggr;1) isotype can kill cells by triggering the complement cascade at the cell surface, resulting in lysis, or through binding C-domain receptors (Fc receptors) on specialised phagocytic and killer cells through ADCC. On another hand, antibodies of the IgG4 isotype (&ggr;4) appear actively to block a response. In the context of the present application this blocking is considered to be an effector function which can be recruited to a chosen target. The binding sites for complement and Fc receptors map to the CH2 domain, sequence variation between CH2 domains of the different isotypes results in different strengths of interaction with complement and Fc receptors. All isotypes except IgE require that the C-domain is correctly glycosylated.
By association of the V-region with a given C-region isotype, an appropriate immune response can be triggered when the antibody binds to antigen. Because the type of immune response is governed by the isotype, artificially-made antibodies can be endowed with appropriate constant regions to be used therapeutically, for example to destroy tumour cells (Hale, G et al., Lancet ii, 1394-1399 (1988)).
If an antibody is to be used in such a way that requires recruitment of natural effector functions, then the antibody (except for the IgE isotype) must be manufactured in eukaryotic cells in order that the protein is glycosylated. Unfortunately, the type and extent of glycosylation varies with eukaryotic cell-type and culture conditions (Borys, M. C. et al., Biotechnology 11, 720-725 (1993)), and this can dramatically shorten their longevity in the circulation as well as adversely influencing recruitment of effector functions. There is the added risk that an inappropriately glycosylated antibody will be immunogenic, limiting the duration of the therapy.
One way of circumventing the need for correctly glycosylated constant regions is to manufacture antibodies comprising at least two different antigen-binding sites. These are known as bispecific antibodies and they can
Holliger Kaspar Philipp
Winter Gregory Paul
Medical Reseach Council
Saunders David
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