Tri- and tetra-valent monospecific antigen-binding proteins

Details Tri- and tetra-valent monospecific antigen-binding proteins Tri- and tetra-valent monospecific antigen-binding proteins

2000-09-18

2003-01-28

McKane, Joseph K. (Department: 1627)

Drug, bio-affecting and body treating compositions

Immunoglobulin, antiserum, antibody, or antibody fragment,...

C424S133100, C424S172100, C424S174100, C530S387300, C530S387700

Reexamination Certificate

active

06511663

ABSTRACT:

The present invention relates to tri- and tetra-valent monospecific antigen-binding proteins and to methods for their production as well as to tri- and tetra-valent ligands for their construction. The invention relates in particular, but not exclusively, to the use of recombinant DNA technology to produce such tri- and tetra-valent monospecific antigen-binding proteins.
There has been much interest in recent years in antibodies and their fragments. It is well known that complete antibody molecules are made up of heavy chain and light chain heterodimers. For instance an IgG molecule comprises four polypeptide chains, two heavy-light chain heterodimers. Each light chain consists of two domains, the N-terminal domain being known as the variable or VL domain and the C-terminal domain being known as the constant or CL domain. Each heavy chain consists of four or five domains, depending on the class of the antibody. The N-terminal domain is known as the variable or VH domain. This is attached at its c-terminal end to the N-terminal end of the next domain, which is known as the first constant or CH1 domain. The next part of each heavy chain is known as the hinge region and this is then followed by the second, third and, in some cases, fourth constant or CH2, CH3 and CH4 domains respectively.
In an assembled antibody, the VL and VH domains associate together to form an antigen binding site. Also, the CL and CH1 domains associate together to keep one heavy chain associated with one light chain. Two heavy-light chain heterodimers associate together partly by interaction of the CH2, CH3 and, if present, CH4 domains of the two heavy chains and partly because of interaction between the hinge regions on the two heavy chains.
Each heavy chain hinge region includes at least one, and often several, cysteine residues. In the assembled antibody, the hinge regions of the heavy chains are aligned so that inter-chain disulphide bonds can be formed between the cysteine residues in the hinge regions, covalently bonding the two heavy-light chain heterodimers together. Thus, fully assembled antibodies are at least bivalent in that they have at least two antigen binding sites.
It has been known for some long time that if the disulphide bonds in an antibody's hinge region are broken by mild reduction, it is possible to produce a monovalent antibody comprising a single heavy-light chain heterodimer.
It has also been known for some long time that treatment of antibodies with certain proteolytic enzymes leads to the production of various antibody fragments. For instance, if an antibody is cleaved close to the N-terminal side of each hinge region, two antigen binding fragments (Fab) and one constant region fragment (Fc) are produced. Each Fab fragment comprises the light chain associated with a truncated heavy chain comprising only the VH and CH1 domains. The Fc portion comprises the remaining domains of the heavy chains held together by the hinge region. Alternatively, the antibody may be cleaved close to the C-terminal side of the hinge. This produces a fragment known as the F(ab′)
2
fragment. This essentially comprises two Fab fragments but with the CH1 domains still attached to the hinge regions. Thus, the F(ab′)
2
fragment is a bivalent fragment having the two antigen binding sites linked together by the hinge region. The F(ab′)
2
fragment can be cleaved by reduction to produce a monovalent Fab′ fragment. This can be regarded as being a Fab fragment having on it a hinge region.
It has also proved to be possible, by careful control of digestion conditions, to cleave an antibody between the VL and CL and between the VH and CH1 domains. This gives rise to two fragments known as Fv fragments. Each FV fragment comprises a VL and a VH domain associated with one another. Each Fv fragment is monovalent for antigen binding.
Studies of the amino acid sequence of individual variable domains has shown that there are three areas in each variable domain where the sequence varies considerably. These areas have been termed hypervariable regions or complementarity determining regions (CDRs). The location of these CDRs has been published [Kabat, E. A. et al., in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA, 1987 and Wu, T. T. and Kabat, E. A., J. Exp. Med., 132, 211-250, 1970].
Structural studies on crystallised Fv fragments and molecular modelling studies have shown that each variable domain consists of three loop regions supported on &bgr;-pleated sheet framework regions. In the case of hapten antigen binding the loop regions appear to form a pocket for receiving the antigen.
There is considerable overlap between the CDRs, as determined by sequence analysis, and the loop regions, as determined by structural analysis. However, it is generally accepted that the CDRs, possibly in combination with some extra residues present in the loop region, are primarily involved in determining the antigen binding specificity of the antibody.
In more recent years, there has been much interest in producing antibodies or their fragments by use of recombinant DNA technology. The patent literature is replete with disclosures in this area. Recombinant DNA technology has been used not only to reproduce natural antibodies but also to produce novel antibodies. For instance, it is now possible to produce chimeric antibodies, wherein the variable domains from one species are linked to constant domains from another species.
It is also possible to produce modified antibodies, in which the residues in the CDRs and, if necessary, a number of other residues in the variable domains have been changed so that a different antigen can be bound. This is a useful procedure in that it allows a specificity from, for instance, a mouse monoclonal antibody (MAb) to be created in a human antibody without altering the essentially human nature of the antibody. This has advantages where it is desired to use the antibody in vivo. A further discussion is given in W0-A-91/09967.
WO-A-90/09195 and WO-A-90/09196 relate to cross-linked antibodies and processes for their preparation. Cross linked antibody conjugates are described which have at least one non-disulphide (S—S) interchain bridge optionally containing a reporter or effector molecule. The bridge may be the residue of a homo- or hetero-functional cross-linking reagent and is located away from the antigen binding domains of the antibody. The antibody conjugates have an enhanced binding capacity, in vivo have good blood clearance and, in the presence of a tumour, high tumour: blood and tumour : bone ratios. The conjugates are of use in the diagnosis and therapy of tumours.
They may be prepared by reaction of a cross-linking reagent with an antibody or a fragment thereof. The cross-linking reagent may react either with thiol groups on the antibody molecules or with the side chains of amino acid residues such as glutamic acid, aspartic acid, lysine or tyrosine residues.
However, we have found that while cross linked antibodies as described in WO-A-90/09195 and WO-A-90/09196 have improved properties over natural immunoglobulins and in particular exhibit highly successful binding to tumour cells and good clearance from the blood, they are subject to high uptake by the kidneys and are retained in this tissue. This creates a toxicity problem, particularly when the antibody is radiolabelled for use in therapy and radioimaging. What is required is therefore an antibody molecule which retains the superior binding and clearance properties of cross-linked antibodies but which is not taken up or retained by kidney tissue and thus avoids kidney toxicity problems.
WO-A-91/03493 relates to bi- or tri-valent multispecific Fab conjugates. The conjugates which are described comprise three or four Fab′ antibody fragments linked together using orthophenylenedimaleimide bridging structures. The disclosed trimeric conjugates comprise either two Fab′ fragments of a first specificity and one Fab′ fragment of a second speci

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