Single domain ligands, receptors comprising said ligands,...

Details Single domain ligands, receptors comprising said ligands,... Single domain ligands, receptors comprising said ligands,...

2000-11-28

2003-04-08

Ketter, James (Department: 1636)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023530

Reexamination Certificate

active

06545142

ABSTRACT:

The present invention relates to single domain ligands derived from molecules in the immunoglobulin (Ig) superfamily, receptors comprising at least one such ligand, methods for cloning, amplifying and expressing DNA sequences encoding such ligands, methods for the use of said DNA sequences in the production of Ig-type molecules and said ligands or receptors, and the use of said ligands or receptors in therapy, diagnosis or catalysis.
A list of references is appended to the end of the description. The documents listed therein are referred to in the description by number, which is given in square brackets [].
The Ig superfamily includes not only the Igs themselves but also such molecules as receptors on lymphoid cells such as T lymphocytes. Immunoglobulins comprise at least one heavy and one light chain covalently bonded together. Each chain is divided into a number of domains. At the N terminal end of each chain is a variable domain. The variable domains on the heavy and light chains fit together to form a binding site designed to receive a particular target molecule. In the case of Igs, the target molecules are antigens. T-cell receptors have two chains of equal size, the &agr; and &bgr; chains, each consisting of two domains. At the N-terminal end of each chain is a variable domain and the variable domains on the &agr; and &bgr; chains are believed to fit together to form a binding site for target molecules, in this case peptides presented by a histocompatibility antigen. The variable domains are so called because antigen. The variable domains are so called because their amino acid sequence vary particularly wide variety of target molecules Much research has been carried out in Ig molecules to determined how the variable domains are produced. It has areas of relatively conserved. The three hypervariable areas are generally known a complementary determining regions (CDRs).
Crystallgraphic studies have shown in each variable domain of an Ig molecule the CDRs are supported on framework areas formed by the areas of conserved sequences. The three CDRs are brought together by the framework areas and, together with the CDRs on the other chain, form a pocket in which the target molecule is received.
Since the advent of recombinant DNA technology, there has been much interest in the use of such technology to clone and express Ig molecules and derivatives thereof. This interest is reflected in the numbers of patent applications and other publications on the subject.
The earliest work on the cloning and expressing of full Igs in the patent literature is EP-A-0 120 694 (Boss). The Boss application also relates to the cloning and expression of chimeric antibodies. Chimeric antibodies are Ig-type molecules in which the variable domains from one Ig are fused to constant domains from another Ig. Usually, the variable domains are derived from an Ig from one species (often a mouse Ig) and the constant domains are derived from an Ig from a different species (often a human Ig).
A later European patent application, EP-A-0 125 023 (Genetech), relates to much the same subject as the Boss application, but also relates to the production by recombinant DNA technology of other variations of Ig-type molecules.
EP-A-0 194 276 (Neuberger) discloses not only chimeric antibodies of the type disclosed in the Boss application but also chimeric antibodies in which some or all of the constant domains have been replaced by non-Ig derived protein sequences. For instance, the heavy chain CH
2
and CH
3
domains may be replaced by protein sequences derived from an enzyme or a protein toxin.
EP-A-0 239 400 (Winter) discloses a different approach to the production of Ig molecules. In this approach, only the CDRs from a first type of Ig are grafted onto a second type of Ig in place of its normal CDRs. The Ig molecule thus produced is predominantly of the second type, since the CDRs form a relatively small part of the whole Ig. However, since the CDRs are the part of the whole Ig. However, since the CDRs are the parts which define the specificity of the Ig, the Ig molecule thus produced has its specificity derived from the first Ig.
Hereinafter, chimeric antibodies, CDR-grafted Igs, the altered antibodies described by Genentech, and fragments, of such Igs such as F(ab′)
2
and Fv fragments are referred to herein as modified antibodies.
One of the main reasons for all the activity in the Ig field using recombinant DNA technology is the desired to use Igs in therapy. It is well known that, using the hybridoma technique developed by Kohler and Milstein, it is possible to produce monoclonal antibodies (MAbs) of almost any specificity. Thus, MAbs directed against cancer antigens have been produced. It is envisaged that these MAbs could be covalently attached or fused to toxins to provide “magic bullets” for use in cancer therapy. MAbs directed against normal tissue or cell surface antigens have also been produced. Labels can be attached to these so that they can be used for in vivo imaging.
The major obstacle to the use of such MAbs in therapy or in vivo diagnosis is that the vast majority of MAbs which are produced are of rodent, in particular mouse, origin. It is very difficult to produce human MAbs. Since most MAbs are derived from non-human species, they are antigenic in humans. Thus, administration of these MAbs to humans generally results in an anti-Ig response being mounted by the human. Such a response can interfere with therapy or diagnosis, for instance by destroying or clearing the antibody quickly, or can cause allergic reactions or immune complex hypersensitivity which has adverse effects on the patient.
The production of modified Igs has been proposed to ensure that the Ig administered to a patient is as “human” as possible, but still retains the appropriate specificity. It is therefore expected that modified Igs will be as effective sa the MAb form which the specificity is derived but at the same time not very antigenic. Thus, is should be possible to use the modified Ig a reasonable number of times in a treatment or diagnosis regime.
At the level of the gene, it is known that heavy chain variable domains are encoded by a “rearranged” gene which is built from three gene segments: an “unrearranged” VH gene (encoding the N-terminal three framework regions, first two complete CDRs and the first part of the third CDR), a diversity (DH)-segment (DH) (encoding the central portion of the third CDR) and a joining segment (JH) (encoding the last part of the third CDR and the fourth framework region). In the maturation of B-cells, the genes rearranged so that each unrearranged VH gene is linked to one DH gene and one JH gene. The rearranged gene corresponds to VH-DH-JH. This rearranged gene is linked to a gene which encodes the constant portion of the Ig chain.
For light chains, the situation is similar, except that for light chains there is no diversity region. Thus light chain variable domains are encoded by an “unrearranged” VL gene and a JL gene. There are two types of light chains, kappa (&kgr;) or lambda (&lgr;), which are built respectively from unrearranged V&kgr; genes and J&kgr; segments, and from unrearranged V&lgr; genes and J&lgr; segments.
Previous work has shown that it is necessary to have two variable domains in association together for efficient binding. For example, the associated heavy and light chain variable domains were shown to contain the antigen binding site [
1
]. This assumption is borne out by X-ray crystallographic studies of crystallised antibody/antigen complexes [
2
-
6
] which show that both the heavy and light chains of the antibody's variable domains contact the antigen. The expectation that association of heavy and light chain variable domains is necessary for efficient antigen binding underlies work to co-secrete these domains from bacteria [
1
], and to link the domains together by a short section of polypeptide as in the single chain antibodies [
8
,
9
].
Binding of isolated heavy and light chains had also been detect

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