# Patent

## Title
Epitope regions of a thyrotrophin (TSH) receptor, uses thereof and antibodies thereto

## Abstract

The present invention is concerned with epitope regions of a thyrotrophin (TSH) receptor, uses thereof and antibodies thereto.

## Background
This application is a Continuation of U.S. application Ser. No. 12/333,714, filed Dec. 12, 2008, which is a Divisional of U.S. application Ser. No. 10/488,074, filed Jun. 17, 2004, which is a National Stage Application of PCT/GB2002/003831, filed Aug. 21, 2002, which claims benefit of Serial No. 0215212.2, filed Jul. 1, 2002 in Great Britain, and also claims benefit of Serial No. 0120649.9, filed Aug. 23, 2001 in Great Britain and which application(s) are incorporated herein by reference. A claim of priority to all, to the extent appropriate is made.


The present invention is concerned with epitope regions of a thyrotrophin (TSH) receptor, uses thereof and antibodies thereto.
Thyrotrophin or thyroid stimulating hormone (TSH) is a pituitary hormone which plays a key role in regulating the function of the thyroid. Its release is stimulated by the hormone TRH formed in the hypothalamus and controls the formation and release of the important thyroid hormones thyroxine (T4) and tri-iodothyronine (T3). On the basis of a feedback mechanism, the thyroid hormone content of the serum controls the release of TSH. The formation of T3 and T4 by the thyroid cells is stimulated by TSH by a procedure in which the TSH released by the pituitary binds to the TSH receptor of the thyroid cell membrane.
In certain pathological conditions, various types of autoantibodies against this TSH receptor can also be formed. Depending on the type of these autoantibodies, either inhibition of the formation and release of T3 and T4 may occur at the TSH receptor owing to the shielding of the TSH molecules, or, on the other hand, these thyroid hormones may be released in an uncontrolled manner because the anti-TSH receptor autoantibodies mimic the action of the TSH and stimulate the synthesis and release of thyroid hormones.
Autoimmune thyroid disease (AMD) is the most common autoimmune disease affecting different populations worldwide. A proportion of patients with AITD, principally those with Graves' disease, have autoantibodies to the TSH receptor substantially as hereinbefore described. The autoantibodies bind to the TSH receptor and usually mimic the actions of TSH, stimulating the thyroid gland to produce high levels of thyroid hormones. These autoantibodies are described as having stimulating activity. In some patients, autoantibodies bind to the TSH receptor but do not stimulate thyroid hormone production and are described as having blocking activity [J Sanders, Y Oda, S-A Roberts, M Maruyama, J Furmaniak, B Rees Smith “Understanding the thyrotrophin receptor function-structure relationship.” Bailliere's Clinical Endocrinology and Metabolism. Ed. T F Davies 1997 11(3): 451-479. Pub. Bailliere Tindall, London].
Measurements of TSH receptor autoantibodies are important in the diagnosis and management of AMTD, particularly Graves' disease. Currently three types of assays are used to measure TSH receptor autoantibodies:

    
    
        (a) competitive binding assays which measure the ability of TSH receptor autoantibodies to inhibit the binding of TSH to preparations of TSH receptor;
        (b) bioassays which measure the ability of TSH receptor autoantibodies to stimulate cells expressing the TSH receptor in culture; and
        (c) immunoprecipitation of TSH receptor preparations with TSH receptor autoantibodies.
    
    


Measurement of TSH receptor autoantibodies using such assays are described in references J Sanders, Y Oda, S-A Roberts, M Maruyama, J Furmaniak, B Rees Smith “Understanding the thyrotrophin receptor function-structure relationship.” Bailliere's Clinical Endocrinology and Metabolism. Ed. T F Davies 1997 11(3): 451-479. Pub. Bailliere Tindall, London, and J Sanders, Y Oda, S Roberts, A Kiddie, T Richards, J Bolton, V McGrath, S Walters, D Jaskolski, J Furmaniak, B Rees Smith “The interaction of TSH receptor autoantibodies with 125I-labelled TSH receptor.” Journal of Clinical Endocrinology and Metabolism 1999 84(10):3797-3802.
There are, however, a number of limitations associated with the use of the above described currently available assays for measuring TSH receptor autoantibodies. The competitive assays of type (a) which are available in different formats are generally sensitive, relatively easy to perform and adaptable for routine use. However, competitive radioreceptor assays known to date for detecting TSH receptor autoantibodies have fundamental disadvantages of a practical nature which can be ascribed to the fact that the binding ability of TSH receptor preparations generally react very sensitively to changes in the receptor or in a biomolecule bound by it. The binding of biomolecules which are peptides or protein in nature, for example hormones or autoantibodies, to receptors is as a rule very complicated in nature, and the specific binding between receptor and bio-molecule is very much more sensitive to structural alterations, in particular of the receptor, than is the case with a usual antigen/antibody binding pair which is the basis of most immunoassays in which receptors are involved.
Attempts to immobilise and/or to label the TSH receptor have as a rule led to structural alterations which have greatly impaired the functionality of the receptor.
As far as bioassays of the type mentioned in (b) are concerned, these tend to be expensive, time-consuming, require highly skilled staff and are essentially unsuitable for routine use.
With respect to the direct immunoprecipitation assays of type (c), currently available such immunoprecipitation assays do not in practice have the required sensitivity for TSH receptor autoantibody detection.
The present invention alleviates the problems hitherto associated with the prior art detection of TSH receptor autoantibodies. More particularly, the present invention provides diagnostic methods and kits for screening for TSH receptor autoantibodies, with improved sensitivity compared to prior art diagnostic methods and kits, and which, if desired, allow the use of one or more competitive binding partners or competitors for a TSH receptor in competitive assays of the type described above. In particular the present invention is concerned with the use of one or more identified epitope regions of TSH receptor in diagnostic methods and kits for screening for TSH receptor autoantibodies.
There is provided by the present invention, therefore, for use in diagnosis or therapy of autoimmune disease associated with an immune reaction to a TSH receptor, a polypeptide sequence comprising part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

(in particular said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; and/or the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments);

    
    


wherein autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes) with said polypeptide sequence, so as to enable said diagnosis or therapy.
More particularly, there is provided by the present invention for use in diagnosis or therapy of autoimmune disease associated with an immune reaction to a TSH receptor, a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more TSH receptor epitopes with which autoantibodies produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

(in particular said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; and/or the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments);

    
    


wherein autoantibodies produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies) with said polypeptide sequence, so as to enable said diagnosis or therapy.
Alternatively, there is provided by the present invention for use in diagnosis or therapy of autoimmune disease associated with an immune reaction to a TSH receptor, a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more TSH receptor epitopes with which lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

(in-particular said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; and/or the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments);

    
    


wherein lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes) with said polypeptide sequence, so as to enable said diagnosis or therapy.
The present invention further provides for use in diagnosis or therapy of autoimmune disease associated with an immune reaction to a TSH receptor, a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

as depicted in any one of the amino acid sequences of any of FIGS. 1, 3, 5 and 7, (in particular said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments; and/or the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments);

    
    


wherein autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes) with said polypeptide sequence, so as to enable said diagnosis or therapy.
More particularly, the present invention further provides for use in diagnosis or therapy of autoimmune disease associated with an immune reaction to a TSH receptor, a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more TSH receptor epitopes with which autoantibodies produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof or variants, analogs or derivatives of such fragments:

    amino acid numbers 22 to 91 of a TSH receptor;
    amino acid numbers 246 to 260 of a TSH receptor;
    amino acid numbers 260 to 363 of a TSH receptor; and
    amino acid numbers 380 to 418 of a TSH receptor;

as depicted in any one of the amino acid sequences of any of FIGS. 1,3, 5 and 7, (in particular said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments; and/or the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments);



wherein autoantibodies produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies) with said polypeptide sequence, so as to enable said diagnosis or therapy.
The present invention further provides for use in diagnosis or therapy of autoimmune disease associated with an immune reaction to a TSH receptor, a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more TSH receptor epitopes with which lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

as depicted in any one of the amino acid sequences of any of FIGS. 1,3, 5 and 7, (in particular said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments; and/or the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments);

    
    


wherein lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes) with said polypeptide sequence, so as to enable said diagnosis or therapy.
More preferably, it is generally preferred that such diagnostic or therapeutic use employs a polypeptide sequence or sequences comprising, consisting of or consisting essentially of the primary structural conformation of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 32 to 41 of a TSH receptor;
        amino acid numbers 36 to 42 of a TSH receptor;
        amino acid numbers 247 to 260 of a TSH receptor;
        amino acid numbers 277 to 296 of a TSH receptor; and
        amino acid numbers 381 to 385 of a TSH receptor;

(in particular said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; and/or the primary structural conformation of amino acid numbers 247 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 247 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments).

    
    


In particular, it is generally preferred according to the present invention that such diagnostic or therapeutic use employs amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments.
In particular, it is generally preferred according to the present invention that such diagnostic or therapeutic use employs amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments.
In particular, it is generally preferred according to the present invention that such diagnostic or therapeutic use employs amino acid numbers 247 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments.
A particularly preferred such diagnostic or therapeutic use according to the present invention, comprises for use in diagnosis or therapy of autoimmune disease associated with an immune reaction to a TSH receptor:

    
    
        (i) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; and
        (ii) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more further TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments;

wherein autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes) with said polypeptide sequences, so as to enable said diagnosis or therapy.

    
    


More particularly, such diagnostic or therapeutic use may comprise:

    
    
        (i) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more TSH receptor epitopes with which autoantibodies produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; and
        (ii) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more further TSH receptor epitopes with which autoantibodies produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments;

wherein autoantibodies produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies) with said polypeptide sequences, so as to enable said diagnosis or therapy.

    
    


Alternatively, such diagnostic or therapeutic use may comprise:

    
    
        (i) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more TSH receptor epitopes with which lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; and
        (ii) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more further TSH receptor epitopes with which lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments;

wherein lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes) with said polypeptide sequences, so as to enable said diagnosis or therapy.

    
    


A particularly preferred diagnostic or therapeutic use according to the present invention, comprises for use in diagnosis or therapy of autoimmune disease associated with an immune reaction to a TSH receptor:

    
    
        (i) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments; and
        (ii) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more filter TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments;

wherein autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes) with said polypeptide sequences, so as to enable said diagnosis or therapy.

    
    


More particularly, such diagnostic or therapeutic use may comprise:

    
    
        (i) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more TSH receptor epitopes with which autoantibodies produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments; and
        (ii) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more flirter TSH receptor epitopes with which autoantibodies produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments;

wherein autoantibodies produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies) with said polypeptide sequences, so as to enable said diagnosis or therapy.

    
    


Alternatively, such diagnostic or therapeutic use may comprise:

    
    
        (i) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more TSH receptor epitopes with which lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments; and
        (ii) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more further TSH receptor epitopes with which lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments;

wherein lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes) with said polypeptide sequences, so as to enable said diagnosis or therapy.

    
    


It may also be further preferred that the above mentioned diagnostic or therapeutic use employing:

    
    
        (i) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; and
        (ii) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more further TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments;

further employs:

        (iii) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more further TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 381 to 385 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 381 to 385 of a TSH receptor, or variants, analogs or derivatives of such fragments.

More particularly, such preferred diagnostic or therapeutic use employs:

        (i) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments;
        (ii) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more further TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments; and
        (iii) a polypeptide sequence comprising, consisting of or consisting essentially of part or all of the primary structural conformation of one or more further TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), said polypeptide sequence comprising, consisting of or consisting essentially of the primary structural conformation of amino acid numbers 381 to 385 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 7, or one or more variants, analogs, derivatives or fragments of amino acid numbers 381 to 385 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 7, or variants, analogs or derivatives of such fragments.
    
    


As will be appreciated from the accompanying Figures, the above mentioned amino acid sequences can be of human, porcine, bovine, canine, feline, mouse, rat or ovine origin, and the specific amino acid sequences in each of the above mentioned species are hereinafter described in greater detail with reference to FIGS. 1, 3, 5, and 7.
There also provided by the present invention one or more TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), said one or more TSH receptor epitopes comprising, consisting of or consisting essentially of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

(in particular amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; or amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments).

    
    


More particularly, there is provided by the present invention one or more TSH receptor epitopes with which autoantibodies produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies), said one or more TSH receptor epitopes comprising, consisting of or consisting essentially of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments:
amino acid numbers 22 to 91 of a TSH receptor;

    
    
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

(in particular amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; or amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments).

    
    


Alternatively, there is provided by the present invention one or more TSH receptor epitopes with which lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes), said one or more TSH receptor epitopes comprising, consisting of or consisting essentially of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

(in particular amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; or amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments).

    
    


The present invention further provides one or more TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), said one or more TSH receptor epitopes comprising, consisting of or consisting essentially of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

as depicted in any one of the amino acid sequences of any of FIGS. 1, 3, 5 and 7, (in particular amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments; or amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments).

    
    


More particularly, the present invention further provides one or more TSH receptor epitopes with which autoantibodies produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies), said one or more TSH receptor epitopes comprising, consisting of or consisting essentially of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

as depicted in any one of the amino acid sequences of any of FIGS. 1, 3, 5 and 7, (in particular amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments; or amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments).

    
    


The present invention further provides one or more TSH receptor epitopes with which lymphocytes produced in response to a TSH receptor interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes), said TSH receptor epitopes comprising, consisting of or consisting essentially of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

as depicted in any one of the amino acid sequences of any of FIGS. 1, 3, 5 and 7, (in particular amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments; or amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments).

    
    


More preferably, it is generally preferred that one or more TSH receptor epitopes comprise one or more of the following, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 32 to 41 of a TSH receptor;
        amino acid numbers 36 to 42 of a TSH receptor;
        amino acid numbers 247 to 260 of a TSH receptor;
        amino acid numbers 277 to 296 of a TSH receptor; and
        amino acid numbers 381 to 385 of a TSH receptor;

(in particular amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; or amino acid numbers 247 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 247 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments).

    
    


A particularly preferred TSH receptor epitope according to the present invention comprises, consists of or consists essentially of amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments, with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes).
A particularly preferred TSH receptor epitope according to the present invention comprises, consists of or consists essentially of amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments, with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes).
A particularly preferred TSH receptor epitope according to the present invention comprises, consists of or consists essentially of amino acid numbers 247 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments, with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes).
There is also provided by the present invention a polypeptide with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes) and which comprises, consists of or consists essentially of part or all of the primary structural conformation of one or more epitopes of a TSH receptor with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), which polypeptide comprises, consists of or consists essentially of the primary structural conformation of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

(in particular amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; and/or amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments), with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), with the exception of a full length TSH receptor.

    
    


More particularly, there is provided by the present invention a polypeptide with which autoantibodies produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies) and which comprises, consists of or consists essentially of part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of a TSH receptor with which autoantibodies produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies), which polypeptide comprises, consists of or consists essentially of the primary structural conformation of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

(in particular amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; and/or amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments), with which autoantibodies produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies), with the exception of a full length TSH receptor.

    
    


Alternatively, there is provided by the present invention a polypeptide with which lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes) and which comprises, consists of or consists essentially of part or all of the primary structural conformation (that is a continuous sequence of amino acid residues) of one or more epitopes of a TSH receptor with which lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes), which polypeptide comprises, consists of or consists essentially of the primary structural conformation of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

(in particular amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments; and/or amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments), with which lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes), with the exception of a full length TSH receptor.

    
    


The present invention further provides a polypeptide with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes) and which comprises, consists of or consists essentially of part or all of the primary structural conformation of one or more epitopes of a TSH receptor with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), which polypeptide comprises, consists of or consists essentially of the primary structural conformation of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

as depicted in any one of the amino acid sequences of any of FIGS. 1, 3, 5 and 7, (in particular amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments; and/or amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments), with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), with the exception of a full length TSH receptor.

    
    


More particularly, the present invention further provides a polypeptide with which autoantibodies produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies) and which comprises, consists of or consists essentially of part or all of the primary structural conformation of one or more epitopes of a TSH receptor with which autoantibodies produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies), which polypeptide comprises, consists of or consists essentially of the primary structural conformation of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor;
        amino acid numbers 246 to 260 of a TSH receptor;
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

as depicted in any one of the amino acid sequences of any of FIGS. 1, 3, 5 and 7, (in particular amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments; and/or amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments), with which autoantibodies produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies), with the exception of a full length TSH receptor.

    
    


The present invention further provides a polypeptide with which lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes) and which comprises, consists of or consists essentially of part or all of the primary structural conformation of one or more epitopes of a TSH receptor with which lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes), which polypeptide comprises, consists of or consists essentially of the primary structural conformation of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof or variants, analogs or derivatives of such fragments:

    
    
        amino acid numbers 22 to 91 of a TSH receptor,
        amino acid numbers 246 to 260 of a TSH receptor,
        amino acid numbers 260 to 363 of a TSH receptor; and
        amino acid numbers 380 to 418 of a TSH receptor;

as depicted in any one of the amino acid sequences of any of FIGS. 1, 3, 5 and 7, (in particular amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments; and/or amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments), with which lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes), with the exception of a full length TSH receptor.

    
    


More preferably, it is generally preferred that a polypeptide according to the present invention can comprise part or all of the primary structural conformation of one or more epitopes of a TSH receptor with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes) and as such comprises, consists of or consists essentially of the primary structural conformation of one or more of the following, or one or more variants, analogs, derivatives or fragments thereof or variants, analogs or derivatives of such fragments, with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes):

    
    
        amino acid numbers 32 to 41 of a TSH receptor,
        amino acid numbers 36 to 42 of a TSH receptor,
        amino acid numbers 247 to 260 of a TSH receptor,
        amino acid numbers 277 to 296 of a TSH receptor; and
        amino acid numbers 381 to 385 of a TSH receptor.
    
    


Preferably a polypeptide according to the present invention comprises, consists of or consists essentially of, amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments.
Preferably a polypeptide according to the present invention comprises, consists of or consists essentially of, amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments.
Preferably a polypeptide according to the present invention comprises, consists of or consists essentially of, amino acid numbers 247 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments.
It is also preferred according to the present invention that there is provided a polypeptide with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), which polypeptide comprises, consists of or consists essentially of:

    
    
        (i) the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments, with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes); and
        (ii) the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments, with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes);

with the exception of a full length TSH receptor.

    
    


More particularly, there is provided by the present invention a polypeptide with which autoantibodies produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies) and which comprises part or all of the primary structural conformation of TSH receptor epitopes with which autoantibodies produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies), which polypeptide comprises, consists of or consists essentially of:

    
    
        (i) the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments, with which autoantibodies produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies); and
        (ii) the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments, with which autoantibodies produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies);

with the exception of a full length TSH receptor.

    
    


Alternatively, there is provided by the present invention a polypeptide with which lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes) and which comprises part or all of the primary structural conformation of TSH receptor epitopes with which lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes), which polypeptide comprises, consists of or consists essentially of:

    
    
        (i) the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments, with which lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes); and
        (ii) the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments, with which lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes);

with the exception of a full length TSH receptor.

    
    


The present invention further provides a polypeptide with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of epitopes of a TSH receptor with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), which polypeptide comprises, consists of or consists essentially of:

    
    
        (i) the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments, with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes); and
        (ii) the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments, with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes);

with the exception of a fill length TSH receptor.

    
    


More particularly, the present invention further provides a polypeptide with which autoantibodies produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies) and which comprises part or all of the primary structural conformation of TSH rector epitopes with which autoantibodies produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies), which polypeptide comprises, consists of or consists essentially of:

    
    
        (i) the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments, with which autoantibodies produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies); and
        (ii) the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments, with which autoantibodies produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies);

with the exception of a full length TSH receptor.

    
    


The present invention further provides a polypeptide with which lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes) and which comprises part or all of the primary structural conformation of TSH receptor epitopes with which lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes), which polypeptide comprises, consists of or consists essentially of:

    
    
        (i) the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments, with which lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes); and
        (ii) the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments, with which lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such lymphocytes);

with the exception of a full length TSH receptor.

    
    


It is also preferred according to the present invention that there is provided a polypeptide with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of TSH receptor epitopes with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), which polypeptide comprises, consists of or consists essentially of:

    
    
        (i) the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor, or variants, analogs or derivatives of such fragments, with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can react (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes);
        (ii) the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor, or variants, analogs or derivatives of such fragments, with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes); and
        (iii) the primary structural conformation of amino acid numbers 381 to 385 of a TSH receptor, or one or more variants, analogs, derivatives or fragments of amino acid numbers 381 to 385 of a TSH receptor, or variants, analogs or derivatives of such fragments, with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes);

with the exception of a full length TSH receptor.

    
    


More particularly, the present invention further provides a polypeptide with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes) and which comprises part or all of the primary structural conformation of epitopes of a TSH receptor with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes), which polypeptide comprises, consists of or consists essentially of:

    
    
        (i) the primary structural conformation of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or one or more variants, analogs, derivatives or fragments of amino acid numbers 277 to 296 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 5, or variants, analogs or derivatives of such fragments, with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes);
        (ii) the primary structural conformation of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or one or more variants, analogs, derivatives or fragments of amino acid numbers 246 to 260 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 3, or variants, analogs or derivatives of such fragments, with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes);
        (iii) the primary structural conformation of amino acid numbers 381 to 385 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 7, or one or more variants, analogs, derivatives or fragments of amino acid numbers 381 to 385 of a TSH receptor as depicted in any one of the amino acid sequences of FIG. 7, or variants, analogs or derivatives of such fragments, with which autoantibodies and/or lymphocytes produced in response to a TSH receptor can interact (suitably under conditions that allow interaction of a TSH receptor with such autoantibodies or lymphocytes);

with the exception of a full length TSH receptor.

    
    


As will be appreciated from the accompanying Figures, such amino acid sequences can be of human, porcine, bovine, canine, feline, mouse, rat or ovine origin, and the specific amino acid sequences in each of the above mentioned species are hereinafter described in greater detail with reference to FIGS. 1, 3, 5, and 7. Suitably, in the case where polypeptides according to the second aspect of the present invention comprise amino acid sequences corresponding to part or all of the primary structural conformation of more than one epitope of a TSH receptor, the respective amino acid sequences corresponding to part or all of the primary structural conformation of respective epitopes may be separated by linker amino acid sequences so as to preferably provide the respective amino acid sequences in a conformation, arrangement or sequence that resembles or substantially resembles a conformation, arrangement or sequence of amino acids as present in an active site of a TSH receptor, and/or can be effective in providing the above referred to respective amino acid sequences of a TSH receptor in a conformation, arrangement or sequence optimal for interaction with autoantibodies and/or lymphocytes as described herein.
Preferred polypeptide sequences and polypeptides according to the present invention comprise, consist of, or consist essentially of, the specifically referred to amino acid numbered sequences of a TSH receptor as respectively shown in any of accompanying FIGS. 1, 3, 5 or 7. As indicated above, however, the present invention also covers “variants”, “analogs”, “derivatives” and “fragments” of specific amino acid sequences described herein and the terms “variants”, “analogs”, “derivatives” and “fragments” as used herein when referring to polypeptide sequences and polypeptides according to the present invention (such as polypeptides having a primary structural conformation of specified amino acids as described herein with reference to the accompanying Figures) can be characterised as polypeptide sequences and polypeptides which retain essentially the same biological function or activity (in terms of autoantibody and/or lymphocyte interaction as described herein) as polypeptide sequences and polypeptides having a primary structural conformation of specified amino acids as described herein with reference to the accompanying Figures. Suitably, variants, analogs, derivatives and fragments, or variants, analogs or derivatives of the fragments as described herein can have a primary structural conformation of amino acids as seen in the accompanying FIGS. in which several or a few (such as 5 to 10, 1 to 5 or 1 to 3) amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions are deletions which do not alter or substantially alter the biological activity or function of polypeptides according to the present invention as specifically described above. Conservative substitutions can be preferred as hereinafter described in greater detail.
More particularly, variants, analogs or derivatives of polypeptides having a primary structural conformation of specified amino acids as described herein with reference to the accompanying Figures may be:

    
    
        (i) ones in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue); or
        (ii) ones in which one or more of the amino acid resides includes a substituent group; or
        (iii) ones which further comprise additional amino acids that can be effective in providing the above referred to amino acid numbers of a TSH receptor that are present in a polypeptide of the present invention in a conformation, arrangement or sequence that resembles or substantially resembles a conformation, arrangement or sequence of amino acids as present in an active site of a TSH receptor, and/or can be effective in providing the above referred to amino acid numbers of a TSH receptor that are present in a polypeptide of the present invention in a conformation, arrangement or sequence optimal for interaction with autoantibodies and/or lymphocytes as described herein.
    
    


Such variants, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
Typically, variants, analogs or derivatives can be those that vary from a reference (such as polypeptides having a primary structural conformation of specified amino acids as described herein with reference to the accompanying Figures) by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids A, V, L and I; among the hydroxyl residues S and T; among the acidic residues D and E; among the amide residues N and Q; among the basic residues K and R; and among the aromatic residues F and Y.
It may be preferred that variants, analogs or derivatives as provided by the present invention are ones which further comprise additional amino acids that can be effective in providing the above referred to amino acid numbers of a TSH receptor that are present in a polypeptide of the present invention in a conformation, arrangement or sequence that resembles or substantially resembles a conformation, arrangement or sequence of amino acids as present in an active site of a TSH receptor, and/or can be effective in providing the above referred to amino acid numbers of a TSH receptor that are present in a polypeptide of the present invention in a conformation, arrangement or sequence optimal for interaction with autoantibodies and/or lymphocytes as described herein.
More particularly, the term “fragment” as used herein denotes a polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of a polypeptide having a primary structural conformation of specified amino acids—as described herein with reference to the accompanying Figures, and variants or derivatives thereof and such fragments may be “free standing”, i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. As will be appreciated, fragments according to the present invention comprise or contain the primary structural conformation of amino acids present in one or more epitopes of a TSH receptor as described herein so as to be capable of interaction with autoantibodies and/or lymphocytes as described herein.
Polypeptides of the present invention, therefore, include polypeptides having a primary structural conformation of specified amino acids as described herein with reference to the accompanying Figures as well as polypeptides (namely variants, analogs and derivatives as referred to above) having at least 70% identity to polypeptides having a primary structural conformation of specified amino acids as described herein with reference to the accompanying Figures, preferably at least 80% identity to the polypeptides having a primary structural conformation of specified amino acids as described herein with reference to the accompanying Figures, and more preferably at least 90% identity to polypeptides having a primary structural conformation of specified amino acids as described herein with reference to the accompanying Figures and still more preferably at least 95% identity to polypeptides having a primary structural conformation of specified amino acids as described herein with reference to the accompanying Figures and also includes fragments of such polypeptides substantially as referred to above.
A polypeptide according to the present invention is suitably obtained by, or is obtainable by, expression of a polynucleotide according to the present invention substantially as hereinafter described. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesisers employing techniques which are well known in the art. A polypeptide according to the present invention so obtained can be advantageous in being free from association with other eukaryotic polypeptides or contaminants which might otherwise be associated therewith in its natural environment.
Polypeptides according to the present invention substantially as herein described can be expressed in various systems generating recombinant proteins. For example, for expression in E. coli, cDNA coding for the appropriate polypeptides according to the present invention can be cloned into a vector, such as pET22, pMEX8, pGEX2T or pQE81L H is or an equivalent. In the case of expression in yeast (for example Saccharomyces cerevisiae or Schizosaccharomyces pombe), vectors such as pYES2, pESP2 or pYES2/CT or an equivalent, can be employed. AcMNPV (Bac-N-Blue) vector or an equivalent can be used for expression in insect cells and pRC/CMV, pcDNA3.1 vectors or an equivalent can be used for expression in mammalian cells, such as Chinese Hamster Ovary (CHO) cells. A polypeptide according to the present invention can be expressed as a discrete protein, or as a fusion protein linked to, for example, glutathione S transferase (GST) or poly histidine linker. For a discrete protein, affinity column chromatography purification using a mouse monoclonal antibody to the relevant part of a polypeptide according to the present invention coupled to a Sepharose particle can be used. If a polypeptide according to the present invention is fused to GST, glutathione Sepharose chromatography purification can be used to isolate the fusion protein. Specific proteases can be used to separate GST from a polypeptide according to the present invention and a second round of glutathione Sepharose chromatography can be used to separate GST from a polypeptide according to the present invention. In the case of peptides linked to poly histidine linker, the purification can be carried out using immobilised metal affinity chromatography.
The present invention further provides a process of preparing a polypeptide substantially as hereinbefore described, which process comprises:

    
    
        (i) providing a host cell substantially as hereinbefore described;
        (ii) growing the host cell; and
        (iii) recovering a polypeptide according to the present invention therefrom.
    
    


Recovery of a polypeptide according to the present invention can typically employ conventional isolation and purification techniques, such as chromatographic separations or immunological separations, known to one of ordinary skill in the art.
In accordance with a further aspect of the present invention, there is provided a polynucleotide comprising:

    
    
        (i) a nucleotide sequence encoding a polypeptide substantially as hereinbefore described;
        (ii) a nucleotide sequence encoding a polypeptide substantially as hereinbefore described, which polypeptide comprises an amino acid sequence or sequences of specified amino acid numbers of a TSH receptor which is or arm defined by reference to any of FIGS. 1, 3, 5 and 7;
        (iii) a nucleotide sequence encoding a polypeptide of (ii), which nucleotide sequence comprises nucleotide bases encoding the above mentioned specified amino acid numbers of a TSH receptor which are defined by reference to any of FIGS. 1, 3, 5, and 7, and which nucleotide bases are defined by reference to any of FIGS. 2, 4, 6 and 8;
        (iv) a nucleotide sequence differing from the sequence of (iii) in codon sequence due to the degeneracy of the genetic code;
        (v) a nucleotide sequence comprising an allelic variation of the sequence of (iii);
        (vi) a nucleotide sequence comprising a fragment of any of the sequences of (i), (ii), (iii), (iv) or (v); or
        (vii) a nucleotide sequence which hybridizes under stringent conditions to any of the sequences of (i), (ii), (iii), (iv), (v) or (vi).
    
    


The nucleotide bases of a polynucleotide according to the present invention, encoding the above mentioned epitope regions of a polypeptide according to the present invention, can be summarised as follows.














Amino Acid Numbers
Nucleotide Numbers









22-91
 64-273



32{circumflex over ( )}1
 94-123



36{circumflex over ( )}2
106-126



246-260
736-780



247-260
739-780



260-363
 778-1089



277-296
829-888



380-418
1138-1254



381-385
1141-1155










Polynucleotides of the present invention may be in the form of DNA, including, for instance, cDNA, synthetic DNA and genomic DNA appropriately obtained by cloning or produced by chemical synthetic techniques or by a combination thereof A preferred embodiment of the present invention preferably comprises cDNA or synthetic DNA.
The coding sequence which encodes a polypeptide according to the present invention may be identical to the coding sequence of a polynucleotide as referred to above in (iii) and defined by reference to any of FIGS. 2, 4, 6 and 8. It also may be a polynucleotide with a different sequence, which, as a result of the redundancy (degeneracy) of the genetic code, encodes a polypeptide according to the present invention.
The present invention further relates to variants of the herein above described polynucleotides which encode for polypeptides having a primary structural conformation of specified amino acids as described herein with reference to the accompanying Figures, variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of the fragments and substantially as hereinbefore described in greater detail. A variant of the polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques.
Among the variants in this regard are variants that differ from the aforementioned polynucleotides by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions, again substantially as hereinbefore described.
Variant polynucleotides according to the present invention are suitably at least 70% identical over their entire length to a polynucleotide encoding polypeptides having a primary structural conformation of specified amino acids as described herein with reference to the accompanying Figures, and polynucleotides which are complementary to, or hybridise to, such polynucleotides. Alternatively, most highly preferred are polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding a polypeptides having a primary structural conformation of specified amino acids as described herein with reference to the accompanying Figures and polynucleotides which are complementary to, or hybridise to, such polynucleotides. In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% identity are especially preferred. Furthermore, those with at least 97% identity are highly preferred among those with at least 95% identity, and among these those with at least 98% identity and at least 99% identity are particularly highly preferred, with at least 99% identity being the more preferred.
Substantially as hereinbefore described the present invention further relates to polynucleotides that hybridise to the herein above-described sequences. In this regard, the present invention especially relates to polynucleotides which hybridise under stringent conditions to the herein above-described polynucleotides. As herein used, the term “stringent conditions” means hybridisation will occur only if there is at least 95% and preferably at least 97% complementary identity between the sequences.
The present invention also relates to vectors, which comprise a polynucleotide or polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
The present invention, therefore, further provides a biologically functional vector system which carries a polynucleotide substantially as hereinbefore described and which is capable of introducing the polynucleotide into the genome of a host organism.
Host cells can be genetically engineered to incorporate polynucleotides and express polypeptides of the present invention and the present invention further provides a host cell which is transformed or transfected with a polynucleotide, or one or more polynucleotides, or a vector system, each substantially as herein described. The appropriate DNA sequence may be inserted into the vector by any of a variety of well-known and routine techniques.
According to a particularly preferred embodiment of the present invention, there is also provided a method of screening for autoantibodies or lymphocytes produced in response to a TSH receptor in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor, said method comprising:

    
    
        (a) providing either (i) said sample of body fluid from said subject or (ii) lymphocytes isolated from said sample;
        (b) contacting said sample or isolated lymphocytes with a polypeptide according to the present invention substantially as hereinbefore described (suitably under conditions that allow interaction of a TSH receptor with autoantibodies or lymphocytes produced in response to a TSH receptor) so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to a TSH receptor, and present in, or isolated from, said sample; and
        (c) monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to a TSH receptor and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or said lymphocytes, in said sample, or isolated from said sample.
    
    


Substantially as described above, a method according to the present invention is suitable for screening for autoantibodies or lymphocytes produced in response to a TSH receptor in a sample of body fluid obtained from a subject. A method according to the present invention can, however, be particularly adapted for use in screening for autoantibodies produced in response to a TSH receptor in a sample of body fluid obtained from a subject substantially as hereinafter described in greater detail.
There is in particular provided by the present invention, therefore, a method of screening for autoantibodies produced in response to a TSH receptor in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor, said method comprising:

    
    
        (a) providing said sample of body fluid from said subject;
        (b) contacting said sample with a polypeptide according to the present invention substantially as hereinbefore described (suitably under conditions that allow interaction of a TSH receptor with autoantibodies produced in response to a TSH receptor) so as to permit said polypeptide to interact with autoantibodies produced in response to a TSH receptor and present in said sample; and
        (c) monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to a TSH receptor and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample.
    
    


A method according to the present invention may typically employ a control, such as a sample of body fluid from a normal subject, in other words a subject known to be without autoimmune disease associated with an immune reaction to a TSH receptor.
A method of screening for autoantibodies to a -TSH receptor according to the present invention may comprise directly monitoring interaction of (i) autoantibodies to a TSH receptor present in the sample of body fluid from the subject and (ii) a polypeptide, as provided by the present invention substantially as hereinbefore described, typically by employing non-competitive sandwich type assay techniques known in the art.
Typically, in a method according to the present invention employing non-competitive techniques, monitoring of the degree of interaction of (i) autoantibodies to a TSH receptor present in the sample and (ii) a polypeptide according to the present invention substantially as hereinbefore described, can comprise providing labelling means either to a polypeptide according to the present invention substantially as hereinbefore described, or to a binding partner for autoantibodies to a TSH receptor, either of which technique would enable monitoring of the above described interaction. For example, a method according to the present invention may comprise directly or indirectly labelling a polypeptide according to the present invention substantially as hereinbefore described; contacting the thus labelled polypeptide with a sample of body fluid being screened for TSH receptor autoantibodies so as to provide a mixture thereof; and adding to the mixture a binding partner for autoantibodies to a TSH receptor (such as an anti-IgG reagent) present in the sample of body fluid, so as to cause precipitation of any complexes of labelled polypeptide and TSH receptor autoantibodies present in the mixture. Alternatively, it may be preferred that a method according to the present invention further comprises adding a labelled binding partner for TSH receptor autoantibodies (such as a labelled anti-IgG reagent, for example protein A or anti-human IgG, or labelled full length TSH receptor or an epitope thereof) to a mixture obtained by contacting (i) a polypeptide according to the present invention substantially as hereinbefore described immobilised to a support and (ii) a sample of body fluid being screened for autoantibodies to a TSH receptor.
It may alternatively be preferred that a method of screening for autoantibodies to a TSH receptor in the sample of body fluid according to the present invention, utilises the principles employed in known competitive assays. For example, a method according to the present invention may employ at least one competitor capable of competing with autoantibodies to a TSH receptor in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.
Typically, a competitor as employed in a competitive assay method according to the present invention may comprise one or more antibodies, which may be natural or partly or wholly synthetically produced. A competitor as employed in the present invention may alternatively comprise any other protein (for example TSH) having a binding domain or region which is capable of competing with autoantibodies to a TSH receptor in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described. Preferably, however, a competitor as employed in the present invention comprises a monoclonal, recombinant or polyclonal antibody (especially a monoclonal antibody), capable of competing with TSH receptor autoantibodies in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.
Typically, therefore, a competitive assay method according to the present invention may further comprise providing at least one competitor, such as a monoclonal or polyclonal antibody, whereby in step (b) of a method as herein described a polypeptide according to the present invention substantially as hereinbefore described can interact with either a competitor, such as a monoclonal or polyclonal antibody, or autoantibodies to a TSH receptor present in said sample.
Typically monitoring in a competitive assay method according to the present invention comprises comparing:

    
    
        (i) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the absence of said sample of body fluid being screened (that is a suspected disease sample), optionally in the presence of a sample of body fluid from a normal subject, typically a subject known to be without autoimmune disease associated with an immune reaction to a TSH receptor; with
        (ii) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the presence of said sample of body fluid being screened.
    
    


Typically, the comparison involves observing a decrease in interaction of a polypeptide according to the present invention substantially as hereinbefore described and the competitor in (ii) compared to (i) so as to provide an indication of the presence of autoantibodies to a TSH receptor in said sample. Typically, the decrease in interaction can be observed by directly or indirectly labelling the competitor and monitoring any change in the interaction of the thus labelled competitor with a polypeptide according to the present invention substantially as hereinbefore described in the absence and in the presence of a sample of body fluid being screened for autoantibodies to a TSH receptor. Suitably a polypeptide according to the present invention substantially as hereinbefore described may be immobilised to facilitate the above mentioned monitoring.
Alternatively, there is also provided by the present invention a method of screening for autoantibodies to a TSH receptor in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor, said method comprising:

    
    
        (a) providing said sample of body fluid from said subject;
        (b) contacting said sample with
        
            (i) a full length TSH receptor (typically a recombinantly obtained full length TSH receptor), and
            (ii) at least one competitor capable of competing with autoantibodies to a TSH receptor in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described,
            
                (suitably under conditions that allow interaction of a TSH receptor with autoantibodies to a TSH receptor), so as to permit said fill length TSH receptor to interact with either autoantibodies to a TSH receptor present in said sample, or said competitor; and
            
            
        
        
        (c) monitoring the interaction of said full length TSH receptor with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to a TSH receptor in said sample.
    
    


The full length TSH receptor can typically be of human, porcine, bovine, canine, feline, mouse, rat or ovine origin and more preferably a recombinantly obtained full length TSH receptor. A competitor for use in such an assay typically comprises a monoclonal or polyclonal antibody (preferably monoclonal) substantially as hereinbefore described.
Suitably a detectable label that can be employed in a method according to the present invention can be selected from the group consisting of enzymic labels, isotopic labels, chemiluminescent labels, fluorescent labels, dyes and the like.
In the case where an isotopic label (such as 125I, 14C, 3H or 35S) is employed, monitoring may therefore comprise measuring radioactivity dependent on binding of a polypeptide according to the present invention substantially as hereinbefore described. Radioactivity is generally measured using a gamma counter, or liquid scintillation counter.
In the case of a method of screening for lymphocytes according to the present invention, it is generally preferred that lymphocytes are initially isolated from a sample of body fluid from a subject using techniques well known to one of ordinary skill in the art, followed by contact with a polypeptide according to the present invention so as to stimulate the proliferation of the isolated lymphocytes. Monitoring of the effect of interaction of a polypeptide according to the present invention and such proliferating lymphocytes, typically employs means known in the art for monitoring such proliferation of lymphocytes.
According to a further particularly preferred embodiment of the present invention, there is provided a kit for screening for autoantibodies or lymphocytes produced in response to a TSH receptor in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor, said kit comprising:

    
    
        (a) a polypeptide according to the present invention substantially as hereinbefore described;
        (b) means for contacting either (i) a sample of body fluid obtained from said subject, or (ii) lymphocytes isolated from a sample of body fluid obtained from said subject, with said polypeptide according to the present invention substantially as hereinbefore described (suitably under conditions that allow interaction of a TSH receptor with autoantibodies or lymphocytes produced in response to a TSH receptor) so as to permit said polypeptide to interact with autoantibodies, or lymphocytes, produced in response to a TSH receptor, and present in, or isolated from, said sample; and
        (c) means for monitoring the degree, or effect, of interaction of said polypeptide with either said autoantibodies, or said lymphocytes, produced in response to a TSH receptor and present in, or isolated from, said sample, thereby providing an indication of the presence of said autoantibodies, or lymphocytes, in said sample or isolated from said sample.
    
    


Substantially as described above, a kit according to the present invention is suitable for screening for autoantibodies or lymphocytes produced in response to a TSH receptor in a sample of body fluid obtained from a subject. A kit according to the present invention can, however, be particularly adapted for use in screening for autoantibodies produced in response to a TSH receptor in a sample of body fluid obtained from a subject substantially as hereinafter described in greater detail.
There is in particular provided by the present invention, therefore, a kit for screening for autoantibodies produced in response to a TSH receptor in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor, said kit comprising:

    
    
        (a) a polypeptide according to the present invention substantially as hereinbefore described;
        (b) means for contacting a sample of body fluid obtained from said subject with said polypeptide according to the present invention substantially as hereinbefore described (suitably under conditions that allow interaction of a TSH receptor with autoantibodies produced in response to a TSH receptor) so as to permit said polypeptide to interact with autoantibodies produced in response to a TSH receptor and present in said sample; and
        (c) means for monitoring the degree of interaction of said polypeptide with said autoantibodies produced in response to a TSH receptor and present in said sample, thereby providing an indication of the presence of said autoantibodies in said sample.
    
    


A kit according to the present invention may typically further comprise control means, such as means for providing a sample of body fluid from a normal subject, in other words a subject known to be without autoimmune disease associated with an immune reaction to a TSH receptor.
A kit for screening for autoantibodies to a TSH receptor according to the present invention may comprise means for directly monitoring interaction of (i) autoantibodies to a TSH receptor present in the sample of body fluid from the subject and (ii) a polypeptide, as provided by the present invention substantially as hereinbefore described, typically comprising non-competitive sandwich type assay means known in the art.
Typically, in a kit according to the present invention comprising non-competitive assay means, means are provided for monitoring the degree of interaction of (i) autoantibodies to a TSH receptor present in the sample and (ii) a polypeptide according to the present invention substantially as hereinbefore described, and can comprise labelling means provided either to a polypeptide according to the present invention substantially as hereinbefore described, or to a binding partner for autoantibodies to a TSH receptor, either of which would enable monitoring of the above described interaction. For example, a kit according to the present invention may comprise means for directly or indirectly labelling a polypeptide according to the present invention substantially as hereinbefore described; means for contacting the thus labelled polypeptide with a sample of body fluid being screened for a TSH receptor autoantibodies so as to provide a mixture thereof; a binding partner for autoantibodies to a TSH receptor (such as an anti-Ig reagent) present in the sample of body fluid; and means for adding the binding partner to the mixture so as to cause precipitation of any complexes of labelled polypeptide and TSH receptor autoantibodies present in the mixture. Alternatively, it may be preferred that a kit according to the present invention further comprises a labelled binding partner for TSH receptor autoantibodies (such as a labelled anti-IgG reagent, for example protein A or anti-human IgG, or labelled full length a TSH receptor or an epitope thereof) and means for adding the labelled binding partner to a mixture obtained by contacting (i) a polypeptide according to the present invention substantially as hereinbefore described immobilised to a support and (ii) a sample of body fluid being screened for autoantibodies to a TSH receptor.
It may alternatively be preferred that a kit for screening for autoantibodies to a TSH receptor in the sample of body fluid according to the present invention, comprises known competitive assay means. For example, a kit according to the present invention may further comprise at least one competitor capable of competing with autoantibodies to a TSH receptor in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.
Typically, a competitor as employed in a competitive assay kit according to the present invention may comprise one or more antibodies, which may be natural or partly or wholly synthetically produced. A competitor as employed in the present invention may alternatively comprise any other protein having a binding domain or region which is capable of competing with autoantibodies to a TSH receptor in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described. Preferably, however, a competitor as employed in the present invention comprises a monoclonal or polyclonal antibody (especially a monoclonal antibody), capable of competing with TSH receptor autoantibodies in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described.
Typically, therefore, a competitive assay kit according to the present invention may further comprise at least one competitor, such as a monoclonal or polyclonal antibody, whereby a polypeptide according to the present invention substantially as hereinbefore described can interact with either a competitor, such as a monoclonal or polyclonal antibody, or autoantibodies to a TSH receptor present in a sample of body fluid being screened.
Typically monitoring means in a competitive assay kit according to the present invention comprise means for comparing:

    
    
        (i) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the absence of said sample of body fluid being screened (that is a suspected disease sample), optionally in the presence of a sample of body fluid from a normal subject, typically a subject known to be without autoimmune disease associated with an immune reaction to a TSH receptor; with
        (ii) interaction of a polypeptide according to the present invention substantially as hereinbefore described and one or more competitors substantially as hereinbefore described (typically a monoclonal or polyclonal antibody), in the presence of said sample of body fluid being screened.
    
    


Typically, the comparison involves observing a decrease in interaction of a polypeptide according to the present invention substantially as hereinbefore described and the competitor in (ii) compared to (i) so as to provide an indication of the presence of autoantibodies to a TSH receptor in said sample. Typically, the decrease in interaction can be observed by directly or indirectly labelling the competitor and monitoring any change in the interaction of the thus labelled competitor with a polypeptide according to the present invention substantially as hereinbefore described in the absence and in the presence of a sample of body fluid being screened for autoantibodies to a TSH receptor. Suitably a polypeptide according to the present invention substantially as hereinbefore described may be immobilised to facilitate the above mentioned monitoring.
Alternatively, there is also provided by the present invention a kit for screening for autoantibodies to a TSH receptor in a sample of body fluid obtained from a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor, said kit comprising:

    
    
        (a) a full length TSH receptor (typically a recombinantly obtained full length TSH receptor);
        (b) at least one competitor capable of competing with autoantibodies to a TSH receptor in the interaction thereof with a polypeptide according to the present invention substantially as hereinbefore described,
        (c) means for contacting said sample of body fluid from said subject, said fall length TSH receptor and said competitor (suitably under conditions that allow interaction of a TSH receptor with autoantibodies to a TSH receptor), so as to permit said full length TSH receptor to interact with either autoantibodies to a TSH receptor present in said sample, or said competitor; and
        (d) means for monitoring the interaction of said full length TSH receptor with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to a TSH receptor in said sample.
    
    


The full length TSH receptor can typically be of human, porcine, bovine, canine, feline, mouse, rat or ovine origin and more preferably a recombinantly obtained full length TSH receptor. A competitor for use in such an assay kit typically comprises a monoclonal or polyclonal antibody (preferably monoclonal) substantially as hereinbefore described.
Suitably a detectable label that can be employed in a kit according to the present invention can be selected from the group consisting of enzymic labels, isotopic labels, chemiluminescent labels, fluorescent labels, dyes and the like.
In the case where an isotopic label (such as 125I, 14C, 3H or 35S) is employed, monitoring means may therefore comprise means for measuring radioactivity dependent on binding of a polypeptide according to the present invention substantially as hereinbefore described. Radioactivity is generally measured using a gamma counter, or liquid scintillation counter.
In the case of a kit for screening for lymphocytes according to the present invention, it is generally preferred hat means are provided for initially isolating lymphocytes from a sample of body fluid from a subject, using techniques well known to one of ordinary skill in the art, and means are also provided for contacting a polypeptide according to the present invention with such isolated lymphocytes so as to stimulate proliferation of the latter by the former. Means (again known to one of ordinary skill in the art) for monitoring the effect of interaction of a polypeptide according to the present invention and such proliferating lymphocytes, are also provided in such a kit according to the present invention.
It will be appreciated from the foregoing description that the present invention provides assay methods and kits for detecting autoantibodies (in particular) or lymphocytes produced in response to a TSH receptor in a sample of body fluid substantially as hereinbefore described. The detection of such autoantibodies and/or lymphocytes produced in response to a TSH receptor in the sample of body fluid (or at least the level of such autoantibodies and/or lymphocytes in the sample) is indicative of the presence of autoimmune disease associated with an immune reaction to a TSH receptor in the subject from which the sample was obtained and can, therefore, enable the diagnosis of the likely onset or presence of autoimmune disease associated with an immune reaction to a TSH receptor.
There is, therefore, further provided by the present invention a method of diagnosing the likely onset or presence of autoimmune disease associated with an immune reaction to a TSH receptor in a subject (in particular a human) suspected of suffering from, susceptible to, having or recovering from, autoimmune disease associated with an immune reaction to a TSH receptor, the method comprising detecting autoantibodies or lymphocytes produced in response to a TSH receptor in a sample of body fluid from the subject substantially as hereinbefore described, and whereby the detected autoantibodies and/or lymphocytes can provide a diagnosis of the likely onset or presence of autoimmune disease associated with an immune reaction to a TSH receptor in the subject.
There is still further provided by the present invention a method of delaying or preventing the onset of autoimmune disease associated with an immune reaction to a TSH receptor in an animal subject (in particular a human subject) suspected of suffering from, susceptible to or recovering from autoimmune disease associated with an immune reaction to a TSH receptor, which method comprises initially detecting autoantibodies or lymphocytes indicative of the onset or presence of autoimmune disease associated with an immune reaction to a TSH receptor in a sample of body fluid obtained from the subject substantially as hereinbefore described, thereby providing a diagnosis of the likely onset of autoimmune disease associated with an immune reaction to a TSH receptor in the subject, and thereafter therapeutically treating the subject so as to delay the onset and/or prevent autoimmune disease associated with an immune reaction to a TSH receptor.
A polypeptide according to the present invention substantially as hereinbefore described is particularly suitable for use in the therapeutic treatment of autoimmune disease associated with an immune reaction to a TSH receptor. For example, tolerance can be achieved by administering a polypeptide according to the present invention substantially as hereinbefore described to a subject (in particular a human subject) suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor.
There is, therefore, further provided by the present invention a pharmaceutical composition comprising a polypeptide according to the present invention substantially as hereinbefore described, together with a pharmaceutically acceptable carrier, diluent or excipient therefor, wherein the polypeptide can interact with autoantibodies and/or lymphocytes produced in response to a TSH receptor.
The present invention further provides a polypeptide according to the present invention substantially as hereinbefore described for use in the manufacture of a medicament for the treatment of Graves' disease.
Compositions or medicaments according to the present invention should contain a therapeutic or prophylactic amount of at least one polypeptide according to the present invention in a pharmaceutically-acceptable carrier. The pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivery of the polypeptides to the patient. Sterile water, alcohol, fats, waxes, and inert solids may be used as the carrier. Pharmaceutically-acceptable adjuvants, buffering agents, dispersing agents and the like, may also be incorporated into the pharmaceutical compositions. Such compositions can contain a single polypeptide or may contain two or more polypeptides according to the present invention.
It may be desirable to couple a polypeptide according to the present invention to immunoglobulins, e.g. IgG, or to lymphoid cells from the patient being treated in order to promote tolerance. Such an approach is described in Bradley-Mullen, Activation of Distinct Subsets of T Suppressor Cells with Type III Pneutnococcal Polysaccharide Coupled to Syngeneic Spleen Cells, in: IMMUNOLOGICAL TOLERANCE TO SELF AND NON-SELF, Buttisto et al, eds., Annals N.Y. Acad. Sci. Vol. 392, pp 156-166, 1982. Alternatively, the polypeptides may be modified to maintain or enhance binding to the MHC while reducing or eliminating binding to the associated T-cell receptor. In this way, the modified polypeptides may compete with natural a TSH receptor to inhibit helper T-cell activation and thus inhibit the immune response. In all cases, care should be taken that administration of the pharmaceutical compositions of the present invention ameliorate but do not potentiate the autoimmune response.
Pharmaceutical compositions according to the present invention are useful for parenteral administration. Preferably, the compositions will be administered parenterally, i.e. subcutaneously, intramuscularly, or intravenously. Thus, the invention provides compositions for parenteral administration to a patient, where the compositions comprise a solution or dispersion of the polypeptides in an acceptable carrier, as described above. The concentration of the polypeptides in the pharmaceutical composition can vary widely, i.e. from less than about 0.1% by weight, usually being at least about 1% by weight to as much as 20% by weight or more. Typical pharmaceutical compositions for intramuscular injection would be made up to contain, for example, 1 ml of sterile buffered water and 1 to 100/ug of a purified polypeptide of the present invention. A typical composition for intravenous infusion could be made up to contain 100 to 500 ml of sterile Ringer's solution and 100 to 500 mg of a purified polypeptide of the present invention. Actual methods for preparing parenterally administrable compositions are well known in the art and described in more detail in various sources, including, for example, Remington's Pharmaceutical Science, 15th Edition, Mack Publishing Company, Easton, Pa. (1980).
In addition to using a polypeptide according to the present invention directly in pharmaceutical compositions, it is also possible to use a polypeptide according to the present invention to enhance tolerance to a TSH receptor in a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor, employing the following principles. More particularly, peripheral blood lymphocytes can be collected from the subject in a conventional manner and stimulated by exposure to a polypeptide according to the present invention, as defined above. Usually, other mitogens and growth enhancers will be present, e.g., phytohemagglutinin, interleukin 2, and the like. Proliferating T-helper cells may be isolated and cloned, also under the stimulation of a polypeptide according to the present invention. Clones which continue to proliferate may then be used to prepare therapeutic compositions for the subject. The cloned T-cells may be attenuated, e.g. by exposure to radiation, and administered to the subject in order to induce tolerance. Alternatively, the T-cell receptor or portions thereof may be isolated by conventional protein purification methods from the cloned T-cells and administered to the individual. Such immunotherapy methods are described generally in Sinha et al. (1990) Science 248:1380-1388.
In some cases, after a T-helper cell has been cloned as described above, it may be possible to develop therapeutic peptides from the T-cell receptor, where the peptides would be beneficial for treating a patient population suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor. In such cases, the T-cell receptor gene may be isolated and cloned by conventional techniques and peptides based on the receptor produced by recombinant techniques as described above. The recombinantly-produced peptides may then be incorporated in pharmaceutical compositions as described above.
There is also provided by the present invention a method of cloning lymphocytes produced in response to a TSH receptor, which method comprises:

    
    
        providing a source of lymphocytes;
        contacting the lymphocytes with a polypeptide according to the present invention substantially as hereinbefore described, so as to effect proliferation of said lymphocytes; and
        isolating and cloning the proliferating lymphocytes.
    
    


The present invention also provides the use of cloned lymphocytes prepared as above, in the therapeutic treatment of autoimmune disease associated with an immune reaction to a TSH receptor. There is provided, therefore, a pharmaceutical composition comprising cloned lymphocytes prepared as above, together with a pharmaceutically acceptable carrier, diluent or excipient therefor and the use of such cloned lymphocytes in the manufacture of a medicament for the treatment of autoimmune disease associated with an immune reaction to a TSH receptor, in particular Graves' disease.
There is also provided by the present invention one or more therapeutic agents identified as providing a therapeutic effect by interaction with amino acids comprising part or all of the primary conformation of amino acids of one or more epitopes of a TSH receptor substantially as hereinbefore described, and the present invention further provides one or more therapeutic agents for use in therapeutically interacting with amino acids comprising part or all of the primary conformation of amino acids of one or more epitopes of a TSH receptor substantially as hereinbefore described and as such for use in the therapeutic treatment of an autoimmune disease associated with an immune reaction to a TSH receptor.
There is, therefore, still further provided by the present invention a method of treating autoimmune disease associated with an immune reaction to a TSH receptor in a subject, which method comprises initially detecting autoantibodies or lymphocytes produced in response to a TSH receptor in a sample of body fluid obtained from the subject substantially as hereinbefore described, thereby providing a diagnosis of autoimmune disease in the subject, and administering to the subject a therapeutically effective amount of at least one therapeutic agent effective in the treatment of such autoimmune disease, such as a polypeptide according to the present invention substantially as hereinbefore described.
The present invention also provides a method of treating autoimmune disease associated with an immune reaction to a TSH receptor in a subject (in particular a human subject), which method comprises administering to the subject a therapeutically effective amount of a therapeutic agent identified as providing a therapeutic effect by interaction with amino acids comprising part or all of the primary conformation of amino acids of one or more epitopes of a TSH receptor substantially as hereinbefore described
The amount of therapeutic agent administered will depend on the specific autoimmune disease state being treated, possibly the age of the patient and will ultimately be at the discretion of an attendant physician.
There is still further provided by the present invention, in combination, a kit substantially as hereinbefore described, together with a therapeutically effective amount of at least one therapeutic agent effective in the treatment of autoimmune disease associated with an immune reaction to a TSH receptor substantially as hereinbefore described.
Substantially as hereinbefore described, the sample of body fluid being screened by the present invention will typically comprise blood samples or other fluid blood fractions, such as in particular serum samples or plasma samples, but the sample may in principle be another biological fluid, such as saliva or urine or solubilised tissue extracts, or may be obtained by needle biopsy.
There is still further provided by the present invention a binding partner for a TSH receptor, such as an antibody to a TSH receptor, or a fragment of an antibody to a TSH receptor, which binding partner can interact with one or more epitopes to a TSH receptor substantially as hereinbefore described, in particular amino acid numbers 277 to 296 of a TSH receptor. Suitably, antibodies provided by the present invention can be monoclonal (preferred), recombinant or polyclonal. Typically an antibody, such as a monoclonal antibody, as provided by the present invention is in substantially purified form.
More specifically, a monoclonal antibody as provided by the present invention can comprise any of monoclonal antibodies 3C7, 2B4, 8E2, 18C5, 4D7, 16E5, 17D2, 3B3 and 14D3 or active fragments thereof, as described in the Examples and further illustrated by the accompanying Figures. Antibodies such as 2B4, 8E2, 1I8C5, 4D7, 16E5, 17D2, 3B3 and 14D3, or active fragments thereof, as described in the Examples preferably have a high affinity for a TSH receptor, such as at least about 108 molar ̃1. There is, therefore, further provided by the present invention a monoclonal antibody having an affinity of at least about 10 molar′′1 for one or more epitopes of a TSH receptor and which epitope is provided by any one of the following amino acid sequences of a TSH receptor:

    
    
        amino acids 22 to 91 of a TSH receptor; or
        amino acids 246 to 260 of a TSH receptor;

or more particularly, consists essentially of any one of the following amino acid sequences of a TSH receptor:

        amino acids 36 to 42 of a TSH receptor; or
        amino acids 247 to 260 of a TSH receptor.
    
    


There is also provided by the present invention a monoclonal antibody having an affinity of at least about 108 molar−1 for one or more epitopes of a TSH receptor and which epitope is provided by any one of the following amino acid sequences of a TSH receptor:

    
    
        amino acids 32 to 41 of a TSH receptor; or
        amino acids 277 to 296 of a TSH receptor.
    
    


According to a particularly preferred embodiment of the present invention there is provided a binding partner for a TSH receptor, which binding partner is capable of binding to a TSH receptor so as to stimulate the TSH receptor, which binding partner does not comprise TSH or naturally produced autoantibodies to the TSH receptor.
Preferably the binding partner comprises an antibody, in particular a monoclonal or recombinant (preferably monoclonal) antibody, capable of binding to a TSH receptor so as to stimulate the TSH receptor. Examples of monoclonal antibodies disclosed herein which stimulate a TSH receptor in this way include 4D7, 16E5, 17D2 and 14D3.
In a preferred case the present invention provides a binding partner for a TSH receptor, which binding partner is capable of binding to the TSH receptor so as to stimulate the TSH receptor and which comprises:
an antibody VH domain selected from the group consisting of:


    
    
        VH domains as shown in any one of FIGS. 10, 14, 18, 22, 42, 46 or 50, a VH domain comprising one or more VH CDRs with an amino acid sequence corresponding to a VH CDR as shown in FIG. 10, a VH domain comprising one or more VH CDRs with an amino acid sequence corresponding to a VH CDR as shown in FIG. 14, a VH domain comprising one or more VH CDRs with an amino acid sequence corresponding to a VH CDR as shown in FIG. 18, a VH domain comprising one or more VH CDRs with an amino acid sequence corresponding to a VH CDR as shown in FIG. 22, a VH domain comprising one or more VH CDRs with an amino acid sequence corresponding to a VH CDR as shown in FIG. 42, a VH domain comprising one or more VH CDRs with an amino acid sequence corresponding to a VH CDR as shown in FIG. 46, and a VH domain comprising one or more VH CDRs with an amino acid sequence corresponding to a VH CDR as shown in FIG. 50; and/or

an antibody VL domain selected from the group consisting of:

        VL domains as shown in any one of FIGS. 12, 16, 20, 24, 44, 48 or 52, a VL domain comprising one or more VL CDRs with an amino acid sequence corresponding to a VL CDR as shown in FIG. 12, a VL domain comprising one or more VL CDRs with an amino acid sequence corresponding to a VL CDR as shown in FIG. 16, a VL domain comprising one or more VL CDRs with an amino acid sequence corresponding to a VL CDR as shown in FIG. 20, a VL domain comprising one or more VL CDRs with an amino acid sequence corresponding to a VL CDR as shown in FIG. 24, a VL domain comprising one or more VL CDRs with an amino acid sequence corresponding to a VL CDR as shown in FIG. 44, a VL domain comprising one or more VL CDRs with an amino acid sequence corresponding to a VL CDR as shown in FIG. 48, and a VL domain comprising one or more VL CDRs with an amino acid sequence corresponding to a VL CDR as shown in FIG. 52.
    
    


It may be preferred according to the present invention that a binding partner substantially as hereinbefore described comprises an antibody VH domain substantially as hereinbefore described paired with an antibody VL domain substantially as hereinbefore described to provide an antibody binding site comprising both VH and VL domains for a TSH receptor, although as discussed further an antibody VH domain, or an antibody VL domain, may be independently used to bind a TSH receptor. It will be appreciated, therefore, that a binding partner substantially as hereinbefore described can comprise an antibody VH domain substantially as hereinbefore described in the absence of an antibody VL domain. It will also be appreciated, therefore, that a binding partner substantially as hereinbefore described can comprise an antibody VL domain substantially as hereinbefore described in the absence of an antibody VH domain Alternatively, a binding partner substantially as hereinbefore described can comprise an antibody VH domain paired with an antibody VL domain substantially as hereinbefore described to provide an antibody binding site comprising both VH and VL domains for a TSH receptor.
Preferred embodiments according to the present invention can thus include a binding partner substantially as hereinbefore described comprising an antibody VH domain as shown in FIG. 10 paired with an antibody VL domain as shown in FIG. 12 to provide an antibody binding site, comprising both these VH and VL domains for a TSH receptor; or a binding partner substantially as hereinbefore described comprising an antibody VH domain as shown in FIG. 14 paired with an antibody VL domain as shown in FIG. 16 to provide an antibody binding site, comprising both these VH and VL domains for a TSH receptor; or a binding partner substantially as hereinbefore described comprising an antibody VH domain as shown in FIG. 18 paired with an antibody VL domain as shown in FIG. 20 to provide an antibody binding site comprising both these VH and VL domains for a TSH receptor; or a binding partner substantially as hereinbefore described comprising an antibody VH domain as shown in FIG. 22 paired with an antibody VL domain as shown in FIG. 24 to provide an antibody binding site comprising both VH and VL domains for a TSH receptor; or a binding partner substantially as hereinbefore described comprising an antibody VH domain as shown in FIG. 42 paired with an antibody VL domain as shown in FIG. 44 to provide an antibody binding site comprising both VH and VL domains for a TSH receptor, or a binding partner substantially as hereinbefore described comprising an antibody VH domain as shown in FIG. 46 paired with an antibody VL domain as shown in FIG. 48 to provide an antibody binding site comprising both VH and VL domains for a TSH receptor, or a binding partner substantially as hereinbefore described comprising an antibody VH domain as shown in FIG. 50 paired with an antibody VL domain as shown in FIG. 52 to provide an antibody binding site comprising both VH and VL domains for a TSH receptor.
It is further envisaged according to the present invention that VH domains substantially as hereinbefore described may be paired with VL domains other than those specifically described herein. It is also further envisaged according to the present invention that VL domains substantially as hereinbefore described may be paired with VH domains other than those specifically described herein.
According to an alternative embodiment of the present invention there is provided a binding partner substantially as hereinbefore described for a TSH receptor, which binding partner is capable of binding to the TSH receptor so as to stimulate the TSH receptor and which can comprise:
an antibody VH domain comprising:


    
    
        a VH domain comprising one or more VH CDRs with an amino acid sequence corresponding to a VH CDR as shown in FIG. 10, or a VH domain comprising one or more VH CDRs with an amino acid sequence corresponding to a VH CDR as shown in FIG. 14, or a VH domain comprising one or more VH CDRs with an amino acid sequence corresponding to a VH CDR as shown in FIG. 18, or a VH domain comprising one or more VH CDRs with an amino acid sequence corresponding to a VH CDR as shown in FIG. 22, or a VH domain comprising one or more VH CDRs with an amino acid sequence corresponding to a VH CDR as shown in FIG. 42, or a VH domain comprising one or more VH CDRs with an amino acid sequence corresponding to a VH CDR as shown in FIG. 46, or a VH domain comprising one or more VH CDRs with an amino acid sequence corresponding to a VH CDR as shown in FIG. 50; and/or

an antibody VL domain comprising:

        a VL domain comprising one or more VL CDRS with an amino acid sequence corresponding to a VL CDR as shown in FIG. 12, or a VL domain comprising one or more VL CDRs with an amino acid sequence corresponding to a VL CDR as shown in FIG. 16, or a VL domain comprising one or more VL CDRs with an amino acid sequence corresponding to a VL CDR as shown in FIG. 20, or a VL domain comprising one or more VL CDRs with an amino acid sequence corresponding to a VL CDR as shown in FIG. 24, or a VL domain comprising one or more VL CDRs with an amino acid sequence corresponding to a VL CDR as shown in FIG. 44, or a VL domain comprising one or more VL CDRs with an amino acid sequence corresponding to a VL CDR as shown in FIG. 48, or a VL domain comprising one or more VL CDRs with an amino acid sequence corresponding to a VL CDR as shown in FIG. 52.
    
    


One or more CDRs as referred to above may be taken from the hereinbefore described VH and VL domains and incorporated into a suitable framework. For example, the amino acid sequence of one or more CDRs substantially as hereinbefore described may be incorporated into framework regions of antibodies differing from those specifically disclosed herein, such antibodies thereby incorporating the one or more CDRs and being capable of binding to the TSH receptor, preferably to stimulate the TSH receptor substantially as hereinbefore described. Alternatively, a binding partner according to the present invention may comprise a polypeptide capable of binding to the TSH receptor so as to stimulate the TSH receptor substantially as hereinbefore described and comprising the primary structural conformation of amino acids as represented by one or more CDRs as specifically described herein, optionally together with further amino acids, which further amino acids may enhance the binding affinity of one or more CDRs as described herein for a TSH receptor or may have substantially no role in affecting the binding properties of the polypeptide for a TSH receptor.
Preferably a binding partner according to the present invention includes an antibody. The term “antibody” as used herein describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide having a binding domain which is, or is substantially homologous to, an antibody binding domain. Examples of antibodies are the immunoglobulin isotypes and their isotypic subclasses and fragments which comprise an antigen binding domain such as Fab, scFv or the like.
In particular, fragments of antibodies specifically as herein described form an important aspect of the present invention. In this way, where a binding partner according to the present invention comprises an antibody substantially as hereinbefore described, the antibody may comprise any of the following fragments: (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; (iv) the dAb fragment which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments; and (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site.
Alternatively, in the case where a binding partner according to the present invention comprises an antibody, the antibody may comprise a whole antibody, whereby the antibody includes variable and constant regions, which variable and constant regions can be further illustrated for the antibodies provided by the present invention by reference to any of FIGS. 9 to 24, or 41 to 52.
The present invention, also encompasses variants, analogs and derivatives of the specific binding partners, antibodies, VH domains, VL domains, CDRs and polypeptides disclosed herein, which variants, analogs and derivatives retain the ability to bind to the TSH receptor so as to stimulate the TSH receptor substantially as hereinbefore described. The terms variants, analogs and derivatives are substantially herein before further described in greater detail with respect to polypeptides according to the present invention and what is meant by these terms as hereinbefore described applies also to variants, analogs and derivatives of the specific binding partners according to the present invention.
The present invention also provides a further binding partner capable of binding to the TSH receptor so as to stimulate the TSH receptor substantially as hereinbefore described, and which further binding partner can compete for binding to the TSH receptor with any specific binding partner disclosed herein, which further binding partner does not comprise TSH or autoantibodies to a TSH receptor. In particular this further binding partner may comprise a further antibody having a binding site for an epitope region of a TSH receptor suitably as hereinbefore described, which further antibody is capable of binding to the TSH receptor so as to stimulate the TSH receptor substantially as hereinbefore described and can compete for binding to the TSH receptor with any specific binding partner disclosed herein.
There is also provided by the present invention a polynucleotide comprising:

    
    
        (i) a nucleotide sequence as shown in any of FIGS. 25 to 40, or 53 to 64; or parts of such sequences as shown in FIGS. 26, 28, 30, 32, 34, 36, 38, 40, 54, 56, 58, 60, 62, or 64, encoding an amino acid sequence of an antibody VH domain, an antibody VL domain or CDR as shown in any of FIGS. 10, 12, 14, 16, 18, 20, 22, 24, 42, 44, 46, 48, 50 or 52;
        (ii) a nucleotide sequence encoding a binding partner substantially as hereinbefore described, or encoding an amino acid sequence of an antibody VH domain, an antibody VL domain or CDR of a binding partner substantially as hereinbefore described;
        (iii) a nucleotide sequence encoding a binding partner having a primary structural conformation of amino acids as shown in any of FIGS. 9 to 24 or 41 to 52, or encoding an amino acid sequence of an antibody VH domain, an antibody VL domain or CDR as shown in any of FIGS. 10, 12, 14, 16, 18, 20, 22, 24, 42, 44, 46, 48, 50 or 52;
        (iv) a nucleotide sequence differing from any sequence of (i) in codon sequence due to the degeneracy of the genetic code;
        (v) a nucleotide sequence comprising an allelic variation of any sequence of (i);
        (vi) a nucleotide sequence comprising a fragment of any of the sequences of (i), (ii), (iii), (iv) or (v), and in particular anucleotide sequence comprising a fragment of any of the sequences of (i), (ii), (iii), (iv) or (v) and encoding a Fab fragment, aFd fragment, a Fv fragment, a dAb fragment, an isolated CDR region, F(ab′)2 fragments or a scFv fragment, of a binding partner substantially as hereinbefore described;
        (vii) a nucleotide sequence differing from the any sequence of (i) due to mutation, deletion or substitution of a nucleotide base and encoding a binding partner substantially as hereinbefore described, or encoding an amino acid sequence of an antibody VH domain, an antibody VL domain or CDR of a binding partner substantially as hereinbefore described.
    
    


Variant polynucleotides according to the present invention are suitably at least 70% identical over their entire length to any polynucleotide sequence of (i), most highly preferred are polynucleotides that comprise a region that is at least 80% identical over its entire length to any polynucleotide sequence of (i), polynucleotides at least 90% identical over their entire length to any polynucleotide sequence of (i) are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% identity are especially preferred. What is meant by variants of specific polynucleotide sequences described herein is hereinbefore described in greater detail.
The present invention further provides a biologically functional vector system which carries a polynucleotide substantially as hereinbefore described and which is capable of introducing the polynucleotide into the genome of a host organism.
The present invention also relates to host cells which are transformed with polynucleotides of the invention and the production of binding partners of the invention by recombinant techniques. Host cells can be genetically engineered to incorporate polynucleotides and express binding partners of the present invention.
A binding partner substantially as hereinbefore described may have diagnostic and therapeutic applications, and may advantageously interact or bind with one or more epitope regions of a TSH receptor substantially as hereinbefore described.
Accordingly, a binding partner substantially as hereinbefore described can be employed in screening methods for detecting autoantibodies substantially as hereinbefore described and also in diagnostic methods substantially as hereinbefore described. In this way, binding partners according to the present invention can be employed in place of competitors hitherto described for use in screening methods for detecting autoantibodies substantially as hereinbefore described and also in diagnostic methods substantially as hereinbefore described. Similarly, binding partners according to the present invention can be employed in place of competitors hitherto described for use in kits for use in detecting autoantibodies substantially as hereinbefore described.
The present invention also provides a method of screening for autoantibodies to a TSH receptor in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor, said method comprising:

    
    
        (a) providing said sample of body fluid from said subject;
        (b) contacting said sample with
        
            (i) a full length TSH receptor, one or more epitopes thereof or a polypeptide comprising one or more epitopes of a TSH receptor, and
            (ii) one or more binding partners substantially as hereinbefore described;
            (suitably under conditions that allow interaction of a TSH receptor with autoantibodies produced in response to a TSH receptor) so as to permit said TSH receptor, said one or more epitopes thereof or said polypeptide, to interact with either autoantibodies to a TSH receptor present in said sample, or said one or more binding partners; and
        
        
        (c) monitoring the interaction of said TSH receptor, said one or more epitopes thereof or said polypeptide, with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to a TSH receptor in said sample.
    
    


Preferably, a method according to the present invention as referred to above, further comprises providing labelling means for the one or more binding partners, suitable labelling means being substantially as hereinbefore described.
The present invention also provides a method of screening for autoantibodies produced in response to a TSH receptor in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor, said method comprising:

    
    
        (a) providing said sample of body fluid from said subject;
        (b) contacting said sample with
        
            (i) a full length TSH receptor, one or more epitopes thereof or a polypeptide comprising one or more epitopes of a TSH receptor, and
            (ii) one or more binding members for a TSH receptor;
            (suitably under conditions that allow interaction of a TSH receptor with autoantibodies produced in response to a TSH receptor) so as to permit said TSH receptor, said one or more epitopes thereof or said polypeptide, to interact with either autoantibodies to a TSH receptor present in said sample, or said one of more binding members; and
        
        
        (c) monitoring the interaction of said TSH receptor, said one or more epitopes thereof or said polypeptide, with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to a TSH receptor in said sample;
        
            wherein said one or more binding members are directly or indirectly immobilised to a surface either prior to, or after step (b).
        
        
    
    


Typically the one or more binding members comprise one or more binding partners according to the present invention substantially as hereinbefore described. Suitably, labelling means are provided for the TSH receptor, the one or more epitopes thereof or the polypeptide.
The present invention also provides a kit for screening for autoantibodies to a TSH receptor in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor, said kit comprising:

    
    
        (a) a full length TSH receptor, one or more epitopes thereof or a polypeptide comprising one or more epitopes of a TSH receptor;
        (b) one or more binding partners substantially as hereinbefore described;
        (c) means for contacting said sample of body fluid from said subject, said TSH receptor, said one or more epitopes thereof or said polypeptide, and said one or more binding partners, (suitably under conditions that allow interaction of a TSH receptor with autoantibodies produced in response to a TSH receptor) so as to permit said TSH receptor, said one or more epitopes thereof or said polypeptide, to interact with either autoantibodies to a TSH receptor present in said sample, or said one or more binding partners; and
        (d) means for monitoring the interaction of said TSH receptor, said one or more epitopes thereof or said polypeptide, with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to a TSH receptor in said sample.
    
    


Suitably, a kit as referred to above further comprises labelling means for the one or more binding partners, suitable labelling means being substantially as hereinbefore described.
The present invention also provides a kit for screening for autoantibodies to a TSH receptor in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor, said kit comprising:

    
    
        (a) a full length TSH receptor, one or more epitopes thereof or a polypeptide comprising one or more epitopes of a TSH receptor;
        (b) one or more binding members for a TSH receptor;
        (c) means for contacting said sample of body fluid from said subject, said TSH receptor, said one or more epitopes thereof or said polypeptide, and said one or more binding members, (suitably under conditions that allow interaction of a TSH receptor with autoantibodies produced in response to a TSH receptor) so as to permit said TSH receptor, said one or more epitopes thereof or said polypeptide, to interact with either autoantibodies to a TSH receptor present in said sample, or said one or more binding members;
        (d) means for directly or indirectly immobilising said one or more members to a surface, either before or after contacting said one or more binding members with said sample of body fluid from said subject and said TSH receptor, said one or more epitopes thereof or said polypeptide; and
        (e) means for monitoring the interaction of said TSH receptor, said one or more epitopes thereof or said polypeptide, with said autoantibodies present in said sample, thereby providing an indication of the presence of said autoantibodies to a TSH receptor in said sample.
    
    


Typically the one or more binding members comprise one or more binding partners according to the present invention substantially as hereinbefore described. Suitably, labelling means are provided for the TSH receptor, the one or more epitopes thereof or the polypeptide.
Suitably a method or kit as referred to above can employ a polypeptide or epitope according to the present invention substantially as hereinbefore described.
Substantially as hereinbefore described, in the presence of autoantibodies to the TSH receptor, binding of the TSH receptor to the immobilised binding member or binding partner will be decreased. Such a method and kit for screening for autoantibodies to a TSH receptor can be advantageous in alleviating problems that can be associated with TSH receptor when immobilised to a surface.
A binding partner substantially as hereinbefore described can also be usefully employed in therapy. There is, therefore, further provided by the present invention methods of treatment comprising administration of a specific binding partner substantially as hereinbefore described, pharmaceutical compositions comprising a specific binding partner substantially as hereinbefore described (together with one or more pharmaceutically acceptable carriers, diluents or excipients therefor), and use of a specific binding partner substantially as hereinbefore described in the manufacture of a medicament or composition, in particular a medicament or composition for use in stimulating thyroid tissue, and/or tissue containing a TSH receptor. In particular, a specific binding partner according to the present invention can be employed in oncology, and in particular for use in the diagnosis, management and treatment of thyroid cancer.
Pharmaceutical compositions according to the present invention include those suitable for oral, parenteral and topical administration, although the most suitable route will generally depend upon the condition of a patient and the specific disease being treated. The precise amount of a binding partner substantially as hereinbefore described to be administered to a patient will be the responsibility of an attendant physician, although the dose employed will depend upon a number of factors, including the age and sex of the patient, the specific disease being treated and the route of administration substantially as described above.
There is further provided by the present invention a method of stimulating thyroid tissue, and/or tissue containing a TSH receptor, which method comprises administering to a patient in need of such stimulation a diagnostically or therapeutically effective amount of a binding partner substantially as hereinbefore described.
The present invention also provides in combination, a binding partner substantially as hereinbefore described, together with one or more further agents capable of stimulating thyroid tissue, and/or tissue containing a TSH receptor, for simultaneous, separate or sequential use in stimulating thyroid tissue, and/or tissue containing a TSH receptor. Preferably the one or more further agents comprise recombinant human TSH and/or one or more variants, analogs, derivatives or fragments thereof, or variants, analogs or derivatives of such fragments. Alternatively, the one or more further agents can act independently of binding to the TSH receptor.
The following illustrative explanations are provided to facilitate understanding of certain terms used herein. The explanations are provided as a convenience and are not limitative of the invention
BINDING PARTNER, or BINDING MEMBER, FOR A TSH RECEPTOR, describes a molecule having a binding specificity for a TSH receptor. A binding partner or binding member as described herein may be naturally derived or wholly or partially synthetically produced. Such a binding partner or binding member has a domain or region which specifically binds to and is therefore complementary to one or more epitope regions of a TSH receptor.
C DOMAIN denotes a region of relatively constant amino acid sequence in antibody molecules.
CDR denotes complementary determining regions which are present on both heavy and light chains of antibody molecules and represent regions of most sequence variability. CDRs represent approximately 15 to 20% of variable domains and represent antigen binding sites of an antibody.
FR denotes framework regions and represent the remainder of the variable light domains and variable heavy domains not present in CDRs.
HC denotes part of a heavy chain of an antibody molecule comprising the heavy chain variable domain and the first domain of an IgG constant region.
HOST CELL is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.
IDENTITY, as known in the art, is the relationship between two or more polypeptide sequences, or two or more polynucleotide sequences, as determined by comparing the sequences.
LC denotes a light chain of an antibody molecule.
STIMULATION OF A TSH RECEPTOR by a binding partner or binding member as described herein denotes the ability of the binding partner or binding member to bind to a TSH receptor and to thereby effect, for example, production of cyclic AMP as a result of such binding to the TSH receptor. Such stimulation is analogous to the responses seen on binding of TSHE or TSH receptor autoantibodies, to a TSH receptor and in this way a binding partner or binding member as described herein mimics the effect of TSH, or TSH receptor autoantibody, binding to a TSH receptor.
V DOMAIN denotes a region of highly variable amino acid sequence in antibody molecules.
VH DOMAIN denotes variable regions or domains in heavy chains of antibody molecules.
VL DOMAIN denotes variable regions or domains in light chains of antibody molecules.



The present invention will now be illustrated by the following Figures and Examples, which do not limit the scope of the invention in any way.
FIG. 1 lists amino acids 1 to 200 of (in the following order) human (HTSHR.PRO; SEQ ID NO:1), porcine (PTSHR.PRO; SEQ ID NO:2), bovine (BTSHR.PRO; SEQ ID NO:3), feline (CTSHR.PRO; SEQ ID NO:4), canine (DTSHR.PRO; SEQ ID NO:5), mouse (MTSHR.PRO; SEQ ID NO:6), rat (RTSHR.PRO; SEQ ID NO:7) and ovine (STSHRP.PRO; SEQ ID NO:8) TSH receptors. Majority (SEQ ID NO: 93) represents the consensus sequence.
FIG. 2 lists nucleotide bases 1 to 300 coding for regions of (in the following order) feline (CAT.SEQ; SEQ ID NO:9), bovine (COW.SEQ; SEQ ID NO:10), canine (DOG.SEQ; SEQ ID NO:11), mouse (MOUSE.SEQ; SEQ ID NO:12), porcine (PTSHR.SEQ; SEQ ID NO:13), rat (RAT.SEQ; SEQ ID NO:14), ovine (SHEEP.SEQ; SEQ ID NO:15) and human (HTSHR.SEQ; SEQ ID NO:16) TSH receptors. Majority (SEQ ID NO: 94) represents the consensus sequence.
FIG. 3 lists amino acids 200 to 300 of (in the following order) human (HTSHR.PRO; SEQ ID NO:17), porcine (PTSHR.PRO; SEQ ID NO:18), bovine (BTSHR.PRO; SEQ ID NO:19), feline (CTSHR.PRO; SEQ ID NO:20), canine (DTSHR.PRO; SEQ ID NO:21), mouse (MTSHR.PRO; SEQ ID NO:22), rat (RTSHR.PRO; SEQ ID NO:23) and ovine (STSHRP.PRO; SEQ ID NO:24) TSH receptors. Majority (SEQ ID NO: 95) represents the consensus sequence.
FIG. 4 lists nucleotide bases 700 to 899 coding for regions of (in the following order) feline (CAT.SEQ; SEQ ID NO:25), bovine (COW.SEQ; SEQ ID NO:26), canine (DOG.SEQ; SEQ ID NO:27), mouse (MOUSE.SEQ; SEQ ID NO:28), porcine (PTSHR.SEQ; SEQ ID NO:29), rat (RAT.SEQ; SEQ ID NO:30), ovine (SHEEP.SEQ; SEQ ID NO:31) and human (HTSHR.SEQ; SEQ ID NO:32) TSH receptors. Majority (SEQ ID NO: 96) represents the consensus sequence.
FIG. 5 lists amino acids 250 to 449 of (in the following order) human (HTSHR.PRO; SEQ ID NO:33), porcine (PTSHR.PRO; SEQ ID NO:34), bovine (BTSHR.PRO; SEQ ID NO:35), feline (CTSHR.PRO; SEQ ID NO:36), canine (DTSHR.PRO; SEQ ID NO:37), mouse (MTSHR.PRO; SEQ ID NO:38), rat (RTSHR.PRO; SEQ ID NO:39) and ovine (STSHRP.PRO; SEQ ID NO:40) TSH receptors. Majority (SEQ ID NO: 97) represents the consensus sequence.
FIG. 6 lists nucleotide bases 750 to 1100 coding for regions of (in the following order) feline (CAT.SEQ; SEQ ID NO:41), bovine (COW.SEQ; SEQ ID NO:42), canine (DOG.SEQ; SEQ ID NO:43), mouse (MOUSE.SEQ; SEQ ID NO:44), porcine (PTSHR.SEQ; SEQ ID NO:45), rat (RAT.SEQ; SEQ ID NO:46), ovine (SHEEP.SEQ; SEQ ID NO:47) and human (HTSHR.SEQ; SEQ ID NO:48) TSH receptors. Majority (SEQ ID NO: 98) represents the consensus sequence.
FIG. 7 lists amino acids 350 to 500 of (in the following order) human (HTSHR.PRO; SEQ ID NO:49), porcine (PTSHR.PRO; SEQ ID NO:50), bovine (BTSHR.PRO; SEQ ID NO:51), feline (CTSHR.PRO; SEQ ID NO:52), canine (DTSHR.PRO; SEQ ID NO:53), mouse (MTSHR.PRO; SEQ ID NO:54), rat (RTSHR.PRO; SEQ ID NO:55) and ovine (STSHRP.PRO; SEQ ID NO:56) TSH receptors. Majority (SEQ ID NO: 99) represents the consensus sequence.
FIG. 8 lists nucleotide bases 1100 to 1299 coding for regions of (in the following order) feline (CAT.SEQ; SEQ ID NO:57), bovine (COW.SEQ; SEQ ID NO:58), canine (DOG.SEQ; SEQ ID NO:59), mouse (MOUSE.SEQ; SEQ ID NO:60), porcine (PTSHR.SEQ; SEQ ID NO:61), rat (RAT.SEQ; SEQ ID NO:62), ovine (SHEEP.SEQ; SEQ ID NO:63) and human (HTSHR.SEQ; SEQ ID NO:64) TSH receptors. Majority (SEQ ID NO: 100) represents the consensus sequence.
FIG. 9 lists amino acids of the heavy chain (HC) of 4D7 (SEQ ID NO:65).
FIG. 10 lists amino acids of the heavy chain (HC) of 4D7 (SEQ ID NO:65), showing the variable region or domain (namely amino acid numbers 10 to 115), the CDRs (namely CDR1 amino acid numbers 31 to 35, CDRII amino acid numbers 50 to 66 and CDRIII amino acid numbers 99 to 104) and the constant region or domain (namely amino acid numbers 116 to 200).
FIG. 11 lists amino acids of the light chain (LC) of 4D7 (SEQ ID NO:66).
FIG. 12 lists amino acids of the light chain (LC) of 4D7 (SEQ ID NO:66), showing the variable region or domain (namely amino acid numbers 9 to 111), the CDRs (namely CDR1 amino acid numbers 24 to 38, CDRII amino acid numbers 54 to 60 and CDRIII amino acid numbers 93 to 101) and the constant region or domain (namely amino acids numbers 112 to 211).
FIG. 13 lists amino acids of the heavy chain (HC) of 16E5 (SEQ ID NO:67).
FIG. 14 lists amino acids of the heavy chain (HC) of 16E5 (SEQ ID NO:67), showing the variable region or domain (namely amino acid numbers 9 to 120), the CDRs (namely CDR1 amino acid numbers 31 to 35, CDRII amino acid numbers 50 to 66 and CDRIII amino acid numbers 99 to 109) and the constant region or domain (namely amino acid numbers 121 to 205).
FIG. 15 lists amino acids of the light chain (LC) of 16E5 (SEQ ID NO:68).
FIG. 16 lists amino acids of the light chain (LC) of 16E5 (SEQ ID NO:68), showing the variable region or domain (namely amino acid numbers 9 to 107), the CDRs (namely CDR1 amino acid numbers 24 to 34, CDRII amino acid numbers 50 to 56 and CDRIII amino acid numbers 89 to 97) and the constant region or domain (namely amino acids numbers 108 to 207).
FIG. 17 lists amino acids of the heavy chain (HC) of 17D2 (SEQ ID NO:69).
FIG. 18 lists amino acids of the heavy chain (HC) of 17D2 (SEQ ID NO:69), showing the variable region or domain (namely amino acid numbers 9 to 120), the CDRs (namely CDR1 amino acid numbers 31 to 35, CDRII amino acid numbers 50 to 66 and CDRIII amino acid numbers 99 to 109) and the constant region or domain (namely amino acid numbers 121 to 205).
FIG. 19 lists amino acids of the light chain (LC) of 17D2 (SEQ ID NO:70).
FIG. 20 lists amino acids of the light chain (LC) of 17D2 (SEQ ID NO:70), showing the variable region or domain (namely amino acid numbers 9 to 107), the CDRs (namely CDR1 amino acid numbers 24 to 34, CDRII amino acid numbers 50 to 56 and CDRIII amino acid numbers 89 to 97) and the constant region or domain (namely amino acids numbers 108 to 207).
FIG. 21 lists amino acids of the heavy chain (HC) of 14D3 (SEQ ID NO:71).
FIG. 22 lists amino acids of the heavy chain (HC) of 14D3 (SEQ ID NO:71), showing the variable region or domain (namely amino acid numbers 9 to 120), the CDRs (namely CDR1 amino acid numbers 31 to 35, CDRII amino acid numbers 50 to 66 and CDRIII amino acid numbers 99 to 109) and the constant region or domain (namely amino acid numbers 121 to 205).
FIG. 23 lists amino acids of the light chain (LC) of 14D3 (SEQ ID NO:72).
FIG. 24 lists amino acids of the light chain (LC) of 14D3 (SEQ ID NO:72), showing the variable region or domain (namely amino acid numbers 9 to 107), the CDRs (namely CDR1 amino acid numbers 24 to 34, CDRII amino acid numbers 50 to 56 and CDRIII amino acid numbers 89 to 97) and the constant region or domain (namely amino acids numbers 108 to 207).
FIG. 25 lists nucleotide bases encoding amino acids of the heavy chain (HC) of 4D7 (SEQ ID NO:73) as shown in FIG. 9.
FIG. 26 lists nucleotide bases encoding amino acids of the heavy chain (HC) of 4D7 (SEQ ID NO:73) as shown in FIG. 9, and shows the nucleotide bases encoding the variable region or domain, the CDRs and the constant region or domain as shown in FIG. 10.
FIG. 27 lists nucleotide bases encoding amino acids of the light chain (LC) of 4D7 (SEQ ID NO:74) as shown in FIG. 11.
FIG. 28 lists nucleotide bases encoding amino acids of the light chain (LC) of 4D7 (SEQ ID NO:74) as shown in FIG. 11, and shows the nucleotide bases encoding the variable region or domain, the CDRs and the constant region or domain as shown in FIG. 12.
FIG. 29 lists nucleotide bases encoding amino acids of the heavy chain (HC) of 16E5 (SEQ ID NO:75) as shown in FIG. 13.
FIG. 30 lists nucleotide bases encoding amino acids of the heavy chain (HC) of 16E5 (SEQ ID NO:75) as shown in FIG. 13, and shows the nucleotide bases encoding the variable region or domain, the CDRs and the constant region or domain as shown in FIG. 14.
FIG. 31 lists nucleotide bases encoding amino acids of the light chain (LC) of 16E5 (SEQ ID NO:76) as shown in FIG. 15.
FIG. 32 lists nucleotide bases encoding amino acids of the light chain (LC) of 16E5 (SEQ ID NO:76) as shown in FIG. 15, and shows the nucleotide bases encoding the variable region or domain, the CDRs and the constant region or domain as shown in FIG. 16.
FIG. 33 lists nucleotide bases encoding amino acids of the heavy chain (HC) of 17D2 (SEQ ID NO:77) as shown in FIG. 17.
FIG. 34 lists nucleotide bases encoding amino acids of the heavy chain (HC) of 17D2 (SEQ ID NO:77) as shown in FIG. 17, and shows the nucleotide bases encoding the variable region or domain, the CDRs and the constant region or domain as shown in FIG. 18.
FIG. 35 lists nucleotide bases encoding amino acids of the light chain (LC) of 17D2 (SEQ ID NO:78) as shown in FIG. 19.
FIG. 36 lists nucleotide bases encoding amino acids of the light chain (LC) of 17D2 (SEQ ID NO:78) as shown in FIG. 19, and shows the nucleotide bases encoding the variable region or domain, the CDRs and the constant region or domain as shown in FIG. 20.
FIG. 37 lists nucleotide bases encoding amino acids of the heavy chain (HC) of 14D3 (SEQ ID NO:79) as shown in FIG. 21.
FIG. 38 lists nucleotide bases encoding amino acids of the heavy chain (HC) of 14D3 (SEQ ID NO:79) as shown in FIG. 21, and shows the nucleotide bases encoding the variable region or domain, the CDRs and the constant region or domain as shown in FIG. 22.
FIG. 39 lists nucleotide bases encoding amino acids of the light chain (LC) of 14D3 (SEQ ID NO:80) as shown in FIG. 23.
FIG. 40 lists nucleotide bases encoding amino acids of the light chain (LC) of 14D3 (SEQ ID NO:80) as shown in FIG. 23, and shows the nucleotide bases encoding the variable region or domain, the CDRs and the constant region or domain as shown in FIG. 24.
FIG. 41 lists amino acids of the heavy chain (HC) of 3B3 (SEQ ID NO:81).
FIG. 42 lists amino acids of the heavy chain (HC) of 3B3 (SEQ ID NO:81), showing the variable region or domain (namely amino acid numbers 8 to 112), the CDRs (namely CDR1 amino acid numbers 31 to 35, CDRII amino acid numbers 50 to 66 and CDRIII amino acid numbers 99 to 101) and the constant region or domain (namely amino acid numbers 113 to 196).
FIG. 43 lists amino acids of the light chain (LC) of 3B3 (SEQ ID NO:82).
FIG. 44 lists amino acids of the light chain (LC) of 3B3 (SEQ ID NO:82), showing the variable region or domain (namely amino acid numbers 9 to 111), the CDRs (namely CDR1 amino acid numbers 24 to 38, CDRII amino acid numbers 54 to 60 and CDRIII amino acid numbers 93 to 101) and the constant region or domain (namely amino acids numbers 112 to 211).
FIG. 45 lists amino acids of the heavy chain (HC) of 3C7 (SEQ ID NO:83).
FIG. 46 lists amino acids of the heavy chain (HC) of 3C7 (SEQ ID NO:83), showing the variable region or domain (namely amino acid numbers 10 to 115), the CDRs (namely CDR1 amino acid numbers 31 to 35, CDRII amino acid numbers 50 to 66 and CDRIII amino acid numbers 99 to 104) and the constant region or domain (namely amino acid numbers 116 to 200).
FIG. 47 lists amino acids of the light chain (LC) of 3C7 (SEQ ID NO:84).
FIG. 48 lists amino acids of the light chain (LC) of 3C7 (SEQ ID NO:84), showing the variable region or domain (namely amino acid numbers 9 to 111), the CDRs (namely CDR1 amino acid numbers 24 to 38, CDRII amino acid numbers 54 to 60 and CDRIII amino acid numbers 93 to 101) and the constant region or domain (namely amino acids numbers 112 to 211).
FIG. 49 lists amino acids of the heavy chain (HC) of 2B4 (SEQ ID NO:85).
FIG. 50 lists amino acids of the heavy chain (HC) of 2B4 (SEQ ID NO:85), showing the variable region or domain (namely amino acid numbers 9 to 122), the CDRs (namely CDR1 amino acid numbers 31 to 35, CDRII amino acid numbers 50 to 66 and CDRIII amino acid numbers 99 to 111) and the constant region or domain (namely amino acid numbers 123 to 207).
FIG. 51 lists amino acids of the light chain (LC) of 2B4 (SEQ ID NO:86).
FIG. 52 lists amino acids of the light chain (LC) of 2B4 (SEQ ID NO:86), showing the variable region or domain (namely amino acid numbers 9 to 112), the CDRs (namely CDR1 amino acid numbers 24 to 39, CDRII amino acid numbers 78 to 82 and CDRIII amino acid numbers 94 to 102) and the constant region or domain (namely amino acids numbers 113 to 212).
FIG. 53 lists nucleotide bases encoding amino acids of the heavy chain (HC) of 3B3 (SEQ ID NO:87) as shown in FIG. 41.
FIG. 54 lists nucleotide bases encoding amino acids of the heavy chain (HC) of 3B3 (SEQ ID NO:87) as shown in FIG. 41, and shows the nucleotide bases encoding the variable region or domain, the CDRs and the constant region or domain as shown in FIG. 42.
FIG. 55 lists nucleotide bases encoding amino acids of the light chain (LC) of 3B3 (SEQ ID NO:88) as shown in FIG. 43.
FIG. 56 lists nucleotide bases encoding amino acids of the light chain (LC) of 3B3 (SEQ ID NO:88) as shown in FIG. 43, and shows the nucleotide bases encoding the variable region or domain, the CDRs and the constant region or domain as shown in FIG. 44.
FIG. 57 lists nucleotide bases encoding amino acids of the heavy chain (HC) of 3C7 (SEQ ID NO:89) as shown in FIG. 45.
FIG. 58 lists nucleotide bases encoding amino acids of the heavy chain (HC) of 3C7 (SEQ ID NO:89) as shown in FIG. 45, and shows the nucleotide bases encoding the variable region or domain, the CDRs and the constant region or domain as shown in FIG. 46.
FIG. 59 lists nucleotide bases encoding amino acids of the light chain (LC) of 3C7 (SEQ ID NO:90) as shown in FIG. 47.
FIG. 60 lists nucleotide bases encoding amino acids of the light chain (LC) of 3C7 (SEQ ID NO:90) as shown in FIG. 47, and shows the nucleotide bases encoding the variable region or domain, the CDRs and the constant region or domain as shown in FIG. 48.
FIG. 61 lists nucleotide bases encoding amino acids of the heavy chain (HC) of 2B4 (SEQ ID NO:91) as shown in FIG. 49.
FIG. 62 lists nucleotide bases encoding amino acids of the heavy chain (HC) of 2B4 (SEQ ID NO:91) as shown in FIG. 49, and shows the nucleotide bases encoding the variable region or domain, the CDRs and the constant region or domain as shown in FIG. 50.
FIG. 63 lists nucleotide bases encoding amino acids of the light chain (LC) of 2B4 (SEQ ID NO:92) as shown in FIG. 51.
FIG. 64 lists nucleotide bases encoding amino acids of the light chain (LC) of 2B4 (SEQ ID NO:92) as shown in FIG. 51, and shows the nucleotide bases encoding the variable region or domain, the CDRs and the constant region or domain as shown in FIG. 52.



More specifically, the FIGS. 1 to 8 illustrate the following:
FIG. 1 lists amino acids 1 to 200 of TSH receptors in the above mentioned species, which include the following amino acid sequences employed in the present invention:

    
    
        amino acids 22 to 91 of a TSH receptor,
        amino acids 32 to 41 of a TSH receptor, and
        amino acids 36 to 42 of a TSH receptor.
    
    


FIG. 2 lists nucleotide bases 1 to 300 in the above mentioned species, which include coding regions for the above mentioned amino acid sequences present in FIG. 1.
FIG. 3 lists amino acids 200 to 300 of TSH receptors in the above mentioned species, which include the following amino acid sequences employed in the present invention:

    
    
        amino acids 246 to 260 of a TSH receptor, and
        amino acids 247 to 260 of a TSH receptor.
    
    


FIG. 4 lists nucleotide bases 700 to 899 in the above mentioned species, which include coding regions for the above mentioned amino acid sequences present in FIG. 3.
FIG. 5 lists amino acids 250 to 449 of TSH receptors in the above mentioned species, which include the following amino acid sequences employed in the present invention:

    
    
        amino acids 260 to 363 of a TSH receptor; and
        amino acids 277 to 296 of a TSH receptor.
    
    


FIG. 6 lists nucleotide bases 750 to 1100 in the above mentioned species, which include coding regions for the above mentioned amino acid sequences present in FIG. 5.
FIG. 7 lists amino acids 350 to 500 of TSH receptors in the above mentioned species, which include the following amino acid sequences employed in the present invention:

    
    
        amino acids 380 to 418 of a TSH receptor; and
        amino acids 381 to 385 of a TSH receptor.
    
    


FIG. 8 lists nucleotide bases 1100 to 1299 in the above mentioned species, which include coding regions for the above mentioned amino acid sequences present in FIG. 7.
EXAMPLE 1
(1) Production of Mouse Monoclonal Antibodies to the TSH Receptor
BALB/c mice were immunised with a recombinant, highly purified mature form of the TSH receptor expressed in CHO cells. [Y Oda, J Sanders, M Evans, A Kiddie, A Munkley, C James, T Richards, J Wills, J Furmaniak, B Rees Smith “Epitope analysis of the human thyrotrophin (TSH) receptor using monoclonal antibodies.” Thyroid 2000 10(12): 1051-1059.] Mouse antibodies were also raised by DNA immunization technique with full length human TSHR cDNA cloned in pcDNA3.1. MAbs were cloned using standard techniques and IgGs were purified from culture supernatants by affinity chromatography on Protein A Sepharose. The reactivity of MAbs with the TSH receptor was tested by (a) Western blotting with partially purified receptors, (b) inhibition of TSH binding to the TSH receptor, and (c) immunoprecipitation of 35S-labelled TSH receptors produced in an in vitro transcription/translation system as described in Y Oda, J Sanders, S Roberts, M Maruyama, R Kato, M Perez, V B Peteresen, N Wedlock, J Furmaniak, B Rees Smith “Binding characteristics of antibodies to the TSH receptor.” Journal of Molecular Endocrinology 1998 20: 233-244.
(2) Inhibition of 125I-TSH Binding to the TSH Receptor
The inhibition of 125I-TSH binding to the TSH receptor was analysed in an assay where, 50 μL of detergent solubilised TSH receptor was preincubated with 50 μL of MAb purified as described in step (1) for 15 minutes at room temperature before addition of 100 μL of 125I-TSH (30,000 cpm) followed by incubation at 37° C. for one hour. The complexes of 125I-TSH/TSH receptor were precipitated by addition of 2 mL 16.5% polyethylene glycol and 25 μL healthy blood donor serum, centrifuged at 1500×g for 30 minutes at 4° C., aspirated and the radioactivity of the pellets counted using known techniques.
MAbs termed: 2B4 MAb (at IgG concentration of 5 μg/mL), 8E2 Mab (at IgG concentration of 1 ug/mL;) and 18C5 Mab (at IgG concentration 1 mg/mL) showed 76%, 38% and 91% inhibition of TSH binding, respectively. Fab fragments were produced from 2B4 Mab, 8E2 Mab and 18C5 Mab IgGs by digestion with L-cysteine/papain or pepsin, followed by the separation of Fc and Fab on Protein A column.
(3) Epitope Recognition by MAbs
Western blotting analysis [Y Oda, J Sanders, M Evans, A Kiddie, A Munkley, C James, T Richards, J Wills, J Furmaniak, B Rees Smith “Epitope analysis of the human thyrotrophin (TSH) rector using monoclonal antibodies.” Thyroid 2000 10(12): 1051-1059.] showed that 2B4 MAb bound to an epitope between amino acid (aa) 380 and 418, 8E2 MAb to an epitope between aa 22 and 91 and 18C5 MAb to an epitope between aa 246 and 260 of the TSH receptor sequence. Analysis with overlapping TSH receptor peptides covering these regions [Y Oda, J Sanders, M Evans, A Kiddie, A Munkley, C James, T Richards, J Wills, J Furmnaniak, B Rees Smith “Epitope analysis of the human thyrotrophin (TSH) receptor using monoclonal antibodies.” Thyroid 2000 10(12): 1051-1059.] showed that 2B4 MAb reacted with the aa 381 to 385, 8E2 MAb with the aa 36 to 42 and 18C5 MAb with the aa 247 to 260.
(4) Preparation of 125I-labelled TSH Receptor
Solubilised preparations of TSH receptor were labelled with 125I by way of 125I-labelled MAB (4E31) reactive with the C-terminal end of the TSH receptor prepared as described in J Sanders, Y Oda, S Roberts, A Kiddie, T Richards, J Bolton, V McGrath, S Walters, D Jaskolski, J Furmaniak, B Rees Smith “The interaction of TSH receptor autoantibodies with 125I-labelled TSH receptor.” Journal of Clinical Endocrinology and Metabolism 1999 84(10):3797-3802. Aliquots of 125I-labelled 4E31 F(ab)2 were incubated for 15 minutes at room temperature with solubilised TSH receptor and then used an immunoprecipitation assay as described in step (5).
(5) Inhibition of TSH Receptor Autoantibody (TRAb) Binding to the TSH Receptor by MAbs
The inhibition of TRAb binding to the TSH receptor by MAbs was tested as follows:
10 μL of 125I-labelled TSH receptor (30,000 cpm) prepared in step (4) was preincubated with 20 μL of 2B4 Fab (5 and 10 mg/mL) for 15 minutes at room temperature followed by incubation with 20 μL of TRAb positive patient serum for one hour at room temperature. 50 μL of solid phase Protein A (an anti-human IgG reagent) was then added and incubation continued for one hour at room temperature followed by washing step and centrifugation at 1500×g at 4° C. for 30 minutes; aspiration and counting of the radioactivity of the pellets. Similar experiments were carried out with 8E2 and 18C5 Fabs and the combination of two Fabs together.
RESULTS OF EXAMPLE 1
Results of the inhibition of TRAb binding to the TSH receptor are shown in Table 1.
EXAMPLE 2
Methods
(1) Production of Mouse Monoclonal Antibodies to the TSH Receptor
BALB/C mice were immunised with a recombinant, highly purified mature form of the TSH receptor expressed in CHO cells (Y. Oda, J. Sanders, M. Evans, A. Kiddie, A. Munkley, C. James, T. Richards, J. Wills, J. Furmaniak, B. Rees Smith “Epitope analysis of the human thyrotropin (TSH) receptor using monoclonal antibodies” Thyroid 2000 10(12): 1051-1059). Mouse antibodies were also raised by DNA immunisation techniques with full length human TSHR cDNA cloned in pRC/CM.1. MAbs were cloned using standard techniques and IgGs were purified from culture supernatants by affinity chromatography on Protein A Sepharose.
(Fab)2 fragments were produced from the purified MAb IgGs by digestion with pepsin followed by chromatography on a protein A affinity column as described in Y. Oda, J. Sanders, S. Roberts, M. Maruyama, R P Kato, M. Perez, V B Petersen, N. Wedlock, J. Furmaniak, B. Rees Smith 1998 “Binding characteristics of antibodies to the TSH receptor”. Journal of Molecular Endocrinology 20: 233-244.
Fab fragments were prepared by digestion of the purified MAbs with papain as described in E. Hendry, G. Taylor, F. Grennan-Jones, A. Sullivan, N. Liddy, J. Godfrey, N. Hayakawa, M. Powell J. Furmaniak, B Rees Smith 2001 “X-ray crystal structure of a monoclonal antibody that binds to a major autoantigenic epitope on thyroid peroxidase.” Thyroid 11(12): 1091-1099.
The reactivity of MAbs with the TSH receptor was tested by (a) western blotting with partially purified receptors, (b) inhibition of the TSH binding to the TSH receptor and (c) immunoprecipitation of 35S-labelled TSH receptors produced in an in vitro transcription/translation system as described in Y. Oda, J. Sanders, S. Roberts, M. Maruyama, R. Kato, M. Perez, V B. Petersen, N. Wedlock, J. Furmaniak, B. Rees Smith “Binding characteristics of antibodies to the TSH receptor” Journal of Molecular Endocrinology 1998 20: 233-244.
(2) Inhibition of 125I TSH Binding to the TSH Receptor
(a) PEG Method for Use with Detergent Solubilised TSHR
The inhibition of 125I TSH binding to detergent solubilised TSH receptor was analysed in an assay where 50 μL of MAb purified as described in Methods (1) above was preincubated with receptor for 15 minutes at room temperature before addition of 100 μL of 125I TSH (30,000 cpm) followed by incubation at 37° C. for one hour. The complexes of 125I TSH and TSH receptor were precipitated by addition of 2 mL 16.5% polyethylene glycol and 25 μL healthy blood donor serum, centrifuged at 1500×g for 30 minutes at 4° C., aspirated and the radioactivity of the pellets counted in a gamma counter.
(b) Method Using Tubes Coated with TSHR
In this procedure, plastic tubes are first coated with a MAb such as 4E31 which binds to a part of the TSHR unrelated to TSH or TRAb binding. Detergent solubilised TSHR preparations are then added, captured by the TSHR MAb and then become immobilised on the tube surface in such a way as to be able to bind TSH or TRAb. In particular the MAb 4E31 reactive with the TSHR C terminus (10 μg/mL F(ab)2 preparation in 0.1 M Na2CO3 pH 9.2) was added to plastic tubes (Nune Maxisorp, 200 μL per tube) and coating allowed to proceed overnight 4° C. After washing and post-coating (10 mg/mL bovine serum albumin) the tubes were washed again with assay buffer (10 mM Tris-HCl pH 7.8, 50 mM NaCl, 1 mg/mL bovine serum albumin, 0.1% Triton X-100). 200 μL of a detergent solubilised TSHR preparation was then added and incubated overnight at 4° C. followed by aspiration and washing steps. Thereafter, 20 μL of “start” buffer (10 mM Tris-HCl pH 7.8, 50 mM NaCl, bovine serum albumin 1 mg/mL, 6 mM NaN3, 1% Triton X-100) was added to the TSHR coated tubes followed by 100 μL of purified MAb IgG or patient sera and incubated at room temperature for 2 hours with gentle shaking. After aspiration, the tubes were washed twice with 1 mL of assay buffer before addition of 100 μL of 125I TSH (80,000 cpm) and incubation at room temperature for 20-60 min with shaking. The tubes were then washed twice with 1 mL of assay buffer, aspirated and counted in a gamma counter.
(3) Analysis of Thyroid Stimulating or Blocking Activities of MAbs.
The ability of MAbs to either stimulate the production of cyclic AMP in isolated porcine thyroid cells (thyroid stimulating activity) or to act as TSH antagonists by blocking TSH stimulation of cyclic AMP (blocking activity) was assessed using reagents from Yamasa Corporation, Tokyo, Japan.
In addition the ability of the MAbs to stimulate production of cyclic AMP in Chinese hamster ovary (CHO) cells expressing human TSHR was analysed as described by M. Kita, L. Ahma, P C. Marians, H. Viase, P. Unger, P. N. Graves, T. F. Davies 1999 “Regulation and transfer of a murine model of thyrotropin receptor antibody mediated Graves' disease.” Endocrinology 140: 1392-1398.
(4) Binding of 125I-labelled MAbs to the TSHR and Effect of TRAb
Purified IgG from two of the MAbs that showed thyroid simulating activity (16E5 and 14D3, table 2) were labelled with 125I followed by separation of unincorporated 125I by filtration on Sephadex G-50 as in (4) in Example 1.
Plastic tubes were coated with TSHR preparations as in 2b above. Thereafter, 100 μL of test serum (from healthy blood donors or from patients with Graves' disease) were added and tubes incubated for 2 hours at room temperature with shaking. After this incubation, the tubes were washed 2 times with assay buffer. Then, 100 μL of 125I-labelled 16E5 or 14D3 IgG (30,000 cpm diluted in 20 mM Tris-HCl pH 7.3, 50 mM NaCl, 1 mg/mL bovine serum albumin, 0.1% Triton X-100) was added to the tubes and incubated for 1 hour at room temperature with shaking. The tubes were then washed twice with the same buffer that was used for diluting 125I-labelled MAbs and counted in a gamma counter.
(5) Binding of TSHR to MAb Coated Tubes and Effect of TRAb
Detergent solubilised TSHR preparations (20 μL were incubated for 1 hour at room temperature with 100 μL of test serum and 20 μL of start buffer (2b above). 100 μL of this mixture was then added to plastic tubes coated with TSHR MAb (as in 2b above) and incubated for 1 hour with shaking at room temperature. Then the tubes were aspirated and washed twice (2b above) and 100 μL (30,000 cpm) of 125I-labelled C-terminal TSHR MAb 4E31 F(ab)2 preparation labelled with 125I as in 4 above added. After further incubation for 1 hour at room temperature with shaking, the tubes were aspirated, washed twice and the radioactivity counted with a gamma counter.
Oligonucleotide primers were designed using the sequences as described previously (Kettleborough C. A. et al “Optimization of primers for cloning libraries of mouse immunoglobulin genes using the polymerase chain reaction.” European Journal of Immunology 1993 23:206-211).
Both sense and antisense primers included additional 5′ restriction endonuclease site sequences to facilitate cloning of PCR products. RT-PCR products were cloned into pUC18 DNA prepared by the Qiagen method (Qiagen) and sequenced by the Sanger-Coulson method
RESULTS OF EXAMPLE 2


    (1) Thyroid stimulating activity of the TSHR MAbs is shown in tables 2 and 3. Four of the MAbs (16E5, 14D3, 17D2, and 4D7) were able to stimulate cyclic AMP production in isolated porcine thyroid cells. In addition when Fab fragments from three of these MAbs were tested, all three also stimulated cyclic AMP production (table 2). For comparison a TRAb positive patient serum showed similar levels of stimulation to the MAbs (table 2). Also, TSHR MAb 2B4 which has the ability to inhibit TSH binding to the TSHR strongly did not show thyroid stimulating activity (table 2). Another TSHR MAb and Fab (3B3) did not stimulate cyclic AMP production nor did the Tg MAb Fab 2G2 (table 2).
    
        In a further series of experiments some of the MABS which were able to stimulate porcine thyroid cells (16E5 and 14D3) were tested for their ability to stimulate cyclic AMP production in CHO cells expressing human TSHR (table 3). Similar results were obtained to those observed with porcine thyroid cells.
    
    
    (2) In the presence of sera from healthy blood donors, 125I-labelled 16E5 bound to TSHR coated tubes is in the range from 23 to 35% of total counts added (table 4). In the presence of sera from patients with Graves' disease (all TRAb positive) the binding of 125I-labelled 16E5 was markedly reduced and was in the range from 1.9 to 7.5% (table 4).
    
        This indicated that Graves' disease patient sera with TRAb activity inhibit the binding of TSHR MAb 16E5 to the TSHR. Further experiments with labelled 16E5 are shown in table 5 where a comparison of the effects of Graves' disease patient sera on (a) 125I-labelled 16E5 to TSHR coated tubes and (b) 125I-labelled TSH binding to TSHR coated tubes. Similar experiments to those shown in table 5 were carried out with 125I-labelled TSHR MAb 14D3 and the results are shown in table 6.
        The effects of Graves' disease patient sera on TSHR coated tube binding by 125I-labelled 16E5, 14D3 or TSH were similar with strong inhibition of binding being observed in most cases (tables 5 and 6). In contrast to Graves' disease patient sera, sera from healthy blood donors had little effect on labelled MAb or labelled TSH binding to TSHR coated tubes (tables 5 and 6). Table 7 shows the effect of sera containing autoantibodies other than the TSHR autoantibodies on TSHR coated tube binding by labelled TSH, 16E5 and 14D3. As can be seen from table 7, sera containing autoantibodies to glutamic acid decarboxylase (D1 and D2) or to 21-hydroxylase (A1 and A2) had no effect on TSH or MAb binding. However, the serum G42 from a patient with Graves' disease showed a strong, dose-dependent inhibition of both TSH and MAb binding.
    
    
    (3) As shown in table 8, plastic tubes coated with MAb 16E5 were able to bind TSHR and this binding was inhibited by Graves' sera containing TSHR autoantibodies. In particular, detection of TSHR binding by the 125I-labelled TSHR MAb 4E31 showed that (a) in the presence of sera from healthy blood donors, labelled 4E31 binding ranged from 13.5-17.8% of total cpm added whereas (b) in the presence of Graves' sera, labelled MAb binding ranged from 1.8-4.8% of total cpm added. Similar results were obtained with plastic tubes coated with MAb 14D3 (table 9).


CONCLUSIONS
The results shown in tables 2-9 show:

    
    
        (a) we have produced TSHR MAbs and MAb Fab fragments which can stimulate isolated thyroid cells in a similar way to TRAb in patient sera and in a similar way to TSH. Different MAbs show different degrees of stimulating activity.
        (b) these MAbs can be used instead of labelled TSH in assays for TSHR autoantibodies (TRAb).
        (c) when the MAbs are coated onto plastic surfaces, they can bind TSHR preparations. This binding is inhibited by TRAb in patient sera, thus providing a new type of TRAb assay.
        (d) the ability of the MAbs to stimulate the thyroid means that they are potentially useful as alternatives to TSH in in vivo applications.
    
    


EXAMPLE 3
Inhibition of 125I-16E5 Fab Binding to Solubilised TSH Receptor by TSH Receptor Mabs
Method
The inhibition of 11I-16E5 Fab binding to detergent solubilised TSH receptor was analysed in an assay where 50 μL of Mab IgG (100 μg/ml) purified as described above was incubated with receptor for 30 minutes at room temperature before addition of 100 μL of 125I-16E5 Fab (30,000 cpm) followed by incubation at room temperature for 2 hours. The complexes of 125I-16E5 Fab and TSH receptor were precipitated by addition of 2 ml 16.5% polyethylene glycol and 50 μL healthy blood donor serum, centrifuged at 1500×g for 30 minutes at 4° C., aspirated and the radioactivity of the pellets counted in a gamma counter.
The results are shown in Table 10. From Table 10 it can be seen that Mab 4D7 (which binds to epitope region 246 to 260 and stimulates isolated thyroid cells) quite strongly inhibits labelled 16E5 Fab binding to the TSH receptor (24.2% inhibition). Two other Mabs, 3C7 and 18C5 also quite strongly inhibit 16E5 Fab binding (17 and 15.7% inhibition respectively) and also bind to the epitope region 246 to 260. Weak or no inhibition is observed with the other Mabs. This suggests that epitope region 246 to 260 is involved in 16E5 binding to the TSH receptor. As the other stimulating Mabs 14D3 and 17D2 compete well with 16E5 binding to the TSH receptor as can be seen from Table 10, epitope region 246 to 260 is probably also important for TSH receptor binding by 14D3 and 17D2.







TABLE 1







Inhibition of binding of TRAb in patient serum


(K3) to the TSH receptor by Mab Fabs









Serum K3 1/10










Labelled TSHR




immunoprecipitated (%)
% inhbition













Buffer
17.5
—


2B4 (5 mg/mL)
10.1
42


2B4 (10 mg/mL)
3.9
77.7


18C5 (5 mg/mL)
13.7
21.7


18C5 (10 mg/mL)
9.7
44


8E2 (5 mg/mL)
15.0
14.3


8E2 (10 mg/mL)
13.0
25.7


2B4 + 18C5 (5 mg/mL)
5.7
67.4


18C5 + 8E2 (5 mg/mL)
12.4
29.1


2B4 + 8E2 (5 mg/mL)
8.1
53.7


Unlabelled TSH (2.94 mg/mL)
7.8
55.4


2B4 + 8E2 + 18C5 (3.3 mg/mL)
7.4
57.7













  
    %
    ⁢
    
      
    
    ⁢
    inhibition
  
  =
  
    100
    -
    
      (
      
        
          A
          B
        
        ×
        100
      
      )
    
  




    
    
        where A=125I-TSHR (cpm) immunoprecipitated in the presence of test sera and test Mab Fab as a percentage of total cpm of material added to the tube;
        
            B=125I-TSHR (cpm) immunoprecipitated in the presence of test sera and assay buffer as a percentage of total cpm of material added to the tube
        
        
    
    


The above results show that:

    (1) the sequences of the TSH receptor which are involved in the TSHR binding are also involved in TRAb binding;
    (2) mouse Mab reactive with these sequences can be used effectively to inhibit TRAb binding to the TSH receptor; and
    (3) one or more of the MAbs reactive with one or more of the above TSH receptor sequences can be used to detect and measure TRAb.









TABLE 2







Thyroid stimulating activity of TSHR MAbs


tested using isolated porcine thyroid cells














Stimulation
Inhibition of TSH


Test sample


(%) 1
binding (%) 2, 3














16E5 IgG
200
μg/ml
466
nt



20
μg/ml
332
83.3



2
μg/ml
269
73.6



0.2
μg/ml
157
nt



0.02
μg/ml
52
nt


14D3 IgG
200
μg/ml
557
nt



20
μg/ml
351
76.4



2
μg/ml
323
61.0



0.2
μg/ml
227
nt



0.02
μg/ml
78
nt


17D2 IgG
200
μg/ml
377
nt



20
μg/ml
207
81.3



2
μg/ml
134
73.7


4D7 IgG
200
μg/ml
259
33 4



20
μg/ml
31
nt


3B3 IgGa
200
μg/ml
34
30.7



20
μg/ml
37
 6.1


2B4 IgGa
20
μg/ml
100
nt



2
μg/ml
116
69.9


3C7 Fab
1
mg/ml
348
45.2


4D7 Fab
1
mg/ml
512
48.6


16E5 Fab
200
μg/ml
425
53 5


14D3 Fab
200
μg/ml
648
64 5


17D2 Fab
200
μg/ml
274
45 5


3B3 Fab a
200
μg/ml
42
  66.5 4


2G2 Fab 6
1
mg/ml
55
0 



200
μg/ml
37
0 










TRAb +ve patient
dil 1:2
771
65 7



dil 1:4
530
nt









Pool of healthy blood donor sera
29
 0.6


TRAb negative serum
70
0 





Table 2 footnotes


1 MAb IgG or Fab preparations were diluted in the pool of healthy blood donor sera. Stimulation (%) was calculated as 100 × the ratio of: cyclic AMP produced in the presence of test sample to cyclic AMP produced in the presence of a pool of healthy blood donor sera. A stimulation level of >180% was assessed as positive i.e. this level of stimulation was always greater than that observed by sera from individual healthy blood donors.


2 Inhibition of TSH binding level of >10% is positive


3 Method = coated tube


4 Inhibition tested at 250 μg/ml


5 Inhibition tested at 10 μg/ml


6 2G2 is a MAb reactive with thyroglobulin i.e. unreactive with the TSHR


7 Inhibition with undiluted serum


a 3B3 and 2B4 IgGs act as TSH antagonists i.e. block the ability of TSH to stimulate cyclic AMP production by isolated porcine thyroid cells.


nt = not tested at this concentration















TABLE 3






Stimulation
Inhibition of TSH


Test sample1
(%)2
binding (%)3,4

















16E5 20 μg/mL
850
78.55


14D3 20 μg/mL
908
71.85


2B4 20 μg/mL
111
84.4


TRAb +ve patient
850
65.06


Pool of healthy blood donor sera
100
0





Table 3 footnotes:


1All samples were diluted 1:10 prior to addition to cells.


2Stimulation (%) was calculated as 100 × the ratio of: cyclic AMP produced in the presence of test sample to cyclic AMP produced in the presence of a pool of healthy blood donor sera.


3Inhibition of TSH binding level of >10% is positive.


4Method = coated tubes


5Inhibition tested at 10 μg/mL


6Tested for inhibition undiluted.













TABLE 4







Binding of 125I-labelled Mab 16E5 to TSHR coated


tubes and effect of TRAb in patient sera











125I-16E5 bound to TSHR



Inhibition of TSH
coated tubes (% total


Test Material1
binding (%)2
counts added)












G1
21
5.6


G2
22.7
6.5


G3
24.7
3.5


G4
22.7
6.0


G5
28.1
3.6


G6
29.4
2.5


G7
29.3
5.8


G8
39
1.9


G9
31.9
6.8


G10
34.8
5.4


G11
34.5
3.4


G12
35.3
4.2


G13
35.6
6.2


G14
36.9
2.8


G15
30.3
4.3


G16
35
2.2


G17
47.6
3.9


G18
43
3.4


G19
53.5
3.7


G20
59.2
7.5


G21
58.9
4.9


NPS
<14
27.5


NSF 1
<14
23.3


NSF 2
<14
30.2


NSF 3
<14
29.1


NSF 4
<14
22.8


NSF 5
<14
28.9


NSF 6
<14
31.0


NSF 7
<14
29.2


NSF 8
<14
35.3


NSF 9
<14
26.3


NSF 10
<14
25.2





Table 4 footnotes:


1sera G1-G22 are from patients with Graves' disease


sera NSF 1-NSF 10 are from healthy blood donors


NPS = pool of healthy blood donor sera


2Inhibition of TSH binding >14% is positive; PEG method used













TABLE 5







Effect of Graves' disease patient sera on 125I-16E5 binding and 125I-TSH


binding to TSHR coated tubes












125I-16E5 
Inhibition
125I-TSH




bound to 
of 
bound to




TSHR coated
125I-16E5
TSHR coated 
Inhibition of


Test
tubes (% total 
binding
tubes (% total
125I-TSH


material1
counts added)2
(%)2,3
counts added)4
binding (%)3,4














G23
13.2
44.0
8.9
27.1


G28
5.8
75.4
3.8
68.5


G29
13.3
43.6
8.0
34.4


G30
9.2
61.0
5.3
56.9


G32
11.9
49.6
7.5
38.4


G36
15.5
34.3
10.1
17.5


G38
16.1
31.8
10.0
18.3


G41
17.8
24.6
10.8
11.4


G43
5.9
75.0
4.0
67.2


G44
18.6
21.2
12.4
−ve


G45
5.1
78.4
3.5
71.0


G46
3.8
83.9
2.7
77.9


G47
7.2
69.5
4.3
64.8


G48
6.9
70.8
4.8
60.8


G49
9.1
61.4
6.1
49.6


G50
8.7
63.1
6.3
48.4


G51
11.9
49.6
7.9
35.2


G52
12.3
47.9
7.4
39.0


NSF 4
23.0
2.6
12.5
−ve


NSF 5
25.3
−ve
12.3
−ve


NSF 10
22.4
5.1
12.5
−ve


NSF 16
23.3
1.3
12.0
1.8


NSF 17
24.2
−ve
11.5
5.3


NSF 18
19.9
15.7
11.2
8.0


NSF 20
21.5
8.9
12.3
−ve


NSF 21
23.3
1.3
12.3
−ve


NSF 22
24.5
−ve
12.4
−ve


NSF 26
26.5
−ve
12.8
−ve





Table 5 footnotes:


1Sera G53-G52 are from patients with Graves' disease; sera NSF are from healthy blood donors


2mean binding in the presence of healthy blood donor sera was 23.6% for 125I-16E5


3inhibition of binding was calculated using the formula






  
    %
    ⁢
    
      
    
    ⁢
    inhibition
  
  =
  
    100
    -
    
      (
      
        
          A
          B
        
        ×
        100
      
      )
    
  



where A = binding in the presence of test serum;


B = mean binding in the presence of healthy blood donor sera


4mean binding in the presence of healthy blood donor sera was 12.2% for 125I-TSH













TABLE 6







Binding of 125I-labelled MAb 14D3 to TSHR coated tubes and effect of


TRAb in patient sera












125I-14D3 bound
Inhibition
125I-TSH bound to
Inhibition



to TSHR coated
of 125I-14D3
TSHR coated 
of 125I-TSH


Test
tubes(% total
binding
tubes (% total
binding


material1
counts added)2
(%)2,3
counts added)4
(%)3,4














G23
13.9
20
8.9
26.6


G24
11.3
35
6.9
43.4


G25
14.1
19
7.2
40.5


G26
7.3
58
2.6
78.3


G27
12.3
29.7
7.3
40.1


G28
8.0
54.4
3.8
68.3


G29
13.2
24.4
8.0
34.0


G30
12.5
28.4
5.3
56.6


G31
9.8
44
4.3
64.3


G32
11.4
34.8
7.5
38.0


G33
12.7
27.2
6.1
49.9


G34
10.9
37.5
7.5
37.8


G35
9.8
43.6
4.3
64.6


G36
13.5
22.8
10.1
16.9


G37
11.9
31.6
9.3
23.4


G38
11.3
35.4
10.0
17.6


G39
12.3
29.5
7.9
34.8


G40
9.8
44.0
7.2
40.9


G41
14.0
19.8
10.8
10.7


NSF 4
17.4
0.3
12.5
−ve


NSF 5
16.5
9.1
12.3
−ve


NSF 10
17.6
−ve
12.5
−ve


NSF 16
17.7
−ve
12.0
1.1


NSF 17
17.0
2.7
11.5
4.6


NSF 18
16.6
8.6
11.2
7.3


NSF 20
18.3
−ve
12.3
−ve


NSF 21
16.8
3.6
12.3
−ve


NSF 22
16.3
6.7
12.4
−ve


NSF 26
18.4
−ve
12.8
−ve





Table 6 footnotes:


1Sera G23-41 are from patients with Graves' disease


Sera NSF are from healthy blood donors


2Mean binding in the presence of healthy blood donor sera was 17.4% for 125I-14D3.


3Inhibition of binding was calculated using the formula:






  
    %
    ⁢
    
      
    
    ⁢
    inhibition
  
  =
  
    100
    -
    
      (
      
        
          A
          B
        
        ×
        100
      
      )
    
  



where A = binding in the presence of test serum;


B = mean binding in the presence of healthy blood donor sera


4Mean binding in the presence of healthy blood donor sera was 12.1% for 125I-TSH.













TABLE 7







Effect of sera from various patients on TSHR coated tube binding by


labelled TSH, 16E5 and 14D3









% inhibition of binding to TSHR coated tubes2 using:










Test sample
125I-TSH
125I-16E5
125I-14D3













G42/5
87
71
77


G42/10
82
56
51


G42/20
70
34
24


D1/10
2
3
2


D1/100
−2
3
0


D2/10
1
1
−7


D2/100
−2
0
0


A1/10
−1
3
2


A1/100
−1
3
−1


A2/10
5
3
5


A2/100
1
3
1





Table 7 footnotes:


1Serum G42 is from a patient with Graves' disease;


sera D1 and D2 are from patients with type 1 diabetes mellitus (positive for auto antibodies to glutamic acid decarboxylase);


sera A1 and A2 are from patients with Addison's disease (positive for steroid 21-hydroxylase autoantibodies)


All test samples were diluted in a pool of serum from healthy blood donors and dilution factor shown as /5, /10, /20 or /100


2inhibition of binding was calculated using the formula






  
    %
    ⁢
    
      
    
    ⁢
    inhibition
  
  =
  
    100
    -
    
      (
      
        
          A
          B
        
        ×
        100
      
      )
    
  



where A = binding in the presence of test serum;


B = mean binding in the presence of a pool of healthy blood donor sera













TABLE 8







Effect of patient sera on binding of the TSHR to 16E5 F(ab)2 coated tubes











125I-4E31 labelled TSHR




Test
bound to 16E5 F(ab)2 coated
Inhibition of
Inhibition of TSH


material1
tubes (% total counts added)
binding2
binding (%)3













G43
1.8
91.4
72.3


G44
4.8
77.2
45.1


G45
3.0
85.6
71.8


G46
2.0
90.2
83.8


G47
1.8
91.4
75.3


NSF 10
17.8
14.4
<14


NSF 17
14.8
29.1
<14


NSF 21
13.5
34.9
<14





Table 8 footnotes:


1Sera G43-47 are from patients with Graves' disease;


Sera NSF are from healthy blood donors


2Inhibition of binding was calculated using the formula:






  
    %
    ⁢
    
      
    
    ⁢
    inhibition
  
  =
  
    100
    -
    
      (
      
        
          A
          B
        
        ×
        100
      
      )
    
  



where A = binding 4E31 binding in the presence of test serum;


B = mean labelled 4E31 binding for healthy blood donor sera (15.4%)


3Inhibition of TSH binding >14% is positive;


PEG method used













TABLE 9







Effect of patient sera on binding of the TSHR to 14D3 F(ab)2 coated tubes











125I-4E31 labelled TSHR




Test
bound to 14D3 F(ab)2 coated
Inhibition of
Inhibition of TSH


material1
tubes (% total counts added)
binding2
binding (%)3













Serum A
4.0
70
72


Serum B
6.9
49
40


Serum C
3.0
78
85


Serum D
2.6
81
80


NSF 5
15.1
−12
<14


NSF 17
14.6
−9
<14


NSF 21
12.0
10
<14


NSF 23
11.8
12
<14





Table 9 footnotes:


1 Sera A-D are from patients with Graves' disease;


Sera NSF are from healthy blood donors


2 Inhibition of binding was calculated using the formula:






  
    %
    ⁢
    
      
    
    ⁢
    inhibition
  
  =
  
    100
    -
    
      (
      
        
          A
          B
        
        ×
        100
      
      )
    
  



where A = labelled 4E31 binding in the presence of test serum;


B = mean labelled 4E31 binding for healthy blood donor sera (15.4%)


3Inhibition of TSH binding >14% is positive;


PEG method used













TABLE 10







Inhibition of 125I-16E5 Fab binding to TSHR by TSHR MAbs











IgG (100 μg/ml)
% inhibition
Epitope region (aa)







16E5
70.4
—



17D3
68.8
—



14D3
67.6
—



17D2
69.2
—



2G2
−ve
Thyroglobulin specific



5D6
−ve
22-41



8E2
−ve
22-41



4B5
 7.1
22-41



10C4
−ve
37-56



10D5
−ve
37-71



4D2
−ve
37-71



2E2
−ve
52-71



1D6
−ve
202-221



7B5
−ve
202-221



16B6
−ve
202-221



3C3
11.2
202-236



4B4
−ve
217-236



4E4
−ve
217-236



8D3
−ve
217-236



6D7
−ve
217-236



18C5
15.7
246-260



3C7
17  
246-260



4D7
24.2
246-260



3B3
 8.8
277-296



5B5
−ve
307-326



4E6
−ve
307-326



6E2
−ve
322-341



9C2
−ve
322-341



6B4
−ve
337-356



3E4
−ve
337-371



3F3
−ve
352-371



3B2
−ve
352-371



7C2
−ve
367-386



2B4
−ve
381-385



3E6
 5.4
381-385



8E3
 4.8
381-385



7C4
−ve
381-385



1D5
 4.2
381-385



4E2
−ve
381-385



3D3
−ve
382-401



2C4
−ve
382-401



10C2
−ve
382-401



7E5
−ve
382-401

## Claims

1. A binding partner for a TSH receptor, the binding partner comprising an isolated monoclonal antibody or antigen binding fragment thereof that:
binds to an epitope on the TSH receptor;
has a binding affinity for the TSH receptor of at least about 108 molar−1;
is positive for thyroid stimulating activity at a concentration of 20 μg/ml in a cyclic AMP thyroid cell assay, wherein the binding partner is classified as positive if the thyroid stimulating activity of the binding partner is greater than 180 percent in the cyclic AMP thyroid cell assay, said percent thyroid stimulating activity comprising 100×(ratio of cyclic AMP produced in the thyroid cell assay in the presence of said binding partner to cyclic AMP produced in the thyroid cell assay in the presence of sera pooled from healthy blood donors); and
inhibits TSH binding, the binding partner inhibiting binding of TSH to the TSH receptor in a coated tube binding assay by 61% to 83.3% at a concentration of 2 μg/ml to 20 μg/ml wherein the TSH receptor is immobilized on a surface of the tube and wherein the TSH receptor is from a species selected from a group consisting of porcine, bovine, feline, canine, mouse, rat and ovine and wherein the monoclonal antibody comprises a VH domain comprising the three CDRs from SEQ ID NO:67 paired with a VL domain comprising the three CDRs from SEQ ID NO:68; or a VH domain comprising the three CDRs from SEQ ID NO:69 paired with a VL domain comprising the three CDRs from SEQ ID NO:70; or a VH domain comprising the three CDRs from SEQ ID NO:71 paired with a VL domain comprising the three CDRs from in SEQ ID NO:72.

2. The binding partner according to claim 1, wherein the binding partner is positive for thyroid stimulating activity at a concentration of 2 μg/ml in the cyclic AMP thyroid cell assay.

3. The binding partner according to claim 1, wherein the monoclonal antibody is a recombinant antibody.

4. The binding partner according to claim 1, wherein the antigen binding fragment comprises a Fab fragment, Fv fragment, F(ab')2 fragment, or scFv.

5. The binding partner according to claim 1, wherein the monoclonal antibody comprises a VH domain comprising amino acid residues 9 to 120 of SEQ ID NO:67 and a VL domain comprising amino acid residues 9 to 107 of SEQ ID NO:68.

6. The binding partner according to claim 1, wherein the monoclonal antibody comprises a VH domain comprising amino acid residues 9 to 120 of SEQ ID NO:69 and a VL domain comprising amino acid residues 9 to 107 of SEQ ID NO:70.

7. The binding partner according to claim 1, wherein the monoclonal antibody comprises a VH domain comprising amino acid residues 9 to 120 of SEQ ID NO:71 and a VL domain comprising amino acid residues 9 to 107 of SEQ ID NO:72.

8. The binding partner according to claim 1, wherein the monoclonal antibody comprises:
a VH domain wherein CDR1 comprises amino acid residues 31 to 35 of SEQ ID NO: 67, CDR2 comprises amino acid residues 50 to 66 of SEQ ID NO:67, and CDR3 comprises amino acid residues 99 to 109 of SEQ ID NO:67; and
a VL domain wherein CDR1 comprises amino acid residues 24 to 34 of SEQ ID NO:68, CDR2 comprises amino acid residues 50 to 56 of SEQ ID NO:68, and CDR3 comprises amino acid residues 89 to 97 of SEQ ID NO: 68.

9. The binding partner according to claim 1, wherein the monoclonal antibody comprises:
a VH domain wherein CDR 1 comprises amino acid residues 31 to 35 of SEQ ID NO:69, CDR 2 comprises amino acid residues 50 to 66 of SEQ ID NO:69, and CDR 3 comprises amino acid residues 99 to 109 of SEQ ID NO:69, and
a VL domain wherein CDR1 comprises amino acid residues 24 to 34 of SEQ ID NO:70, CDR2 comprises amino acid residues 50 to 56 of SEQ ID NO:70, and CDR3 comprises amino acid residues 89 to 97 of SEQ ID NO: 70.

10. The binding partner according to claim 1, wherein the monoclonal antibody comprises:
a VH domain wherein CDR1 comprises amino acid residues 31 to 35 of SEQ ID NO: 71, CDR2 comprises amino acid residues 50 to 66 of SEQ ID NO:71, and CDR 3 comprises amino acid residues 99 to 109 of SEQ ID NO:71, and
a VL domain wherein CDR1 comprises amino acid residues 24 to 34 of SEQ ID NO:72, CDR2 comprises amino acid residues 50 to 56 of SEQ ID NO:72, and CDR3 comprises amino acid residues 89 to 97 of SEQ ID NO: 72.

11. A pharmaceutical composition comprising a binding partner according to claim 1 and one or more pharmaceutically acceptable carriers, diluents or excipients therefor.

12. The composition according to claim 11, further comprising recombinant human TSH.

13. A kit for screening for autoantibodies to a TSH receptor in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor, said kit comprising:
(a) a soluble or solubilized TSH receptor comprising the amino acid sequence of amino acid residues 47 to 61 of SEQ ID NO:17 or SEQ ID NO:22; and
(b) one or more binding partners, wherein the binding partner comprising an isolated monoclonal antibody or antigen binding fragment thereof that:
binds to an epitope on the TSH receptor wherein the TSH receptor is from a species selected from a group consisting of porcine, bovine, feline, canine, mouse, rat and ovine;
has a binding affinity for the TSH receptor of at least about 108 molar−1;
is positive for thyroid stimulating activity at a concentration of 20 μg/ml in a cyclic AMP thyroid cell assay and wherein the binding partner is classified as positive if the thyroid stimulating activity of the binding partner is greater than 180 percent in the cyclic AMP thyroid cell assay, said percent thyroid stimulating activity comprising 100×(ratio of cyclic AMP produced in the thyroid cell assay in the presence of said binding partner to cyclic AMP produced in the thyroid cell assay in the presence of sera pooled from healthy blood donors); and
inhibits TSH binding, the binding partner inhibiting binding of TSH to the TSH receptor in a coated tube binding assay by 61% to 83.3% at a concentration of 2 μg/ml to 20 μg/ml wherein the TSH receptor is immobilized on a surface of the tube and wherein the monoclonal antibody comprises the VH domain as shown in SEQ ID NO:67 paired with the VL domain as shown in SEQ ID NO:68; or the VH domain as shown in SEQ ID NO:69 paired with the VL domain as shown in SEQ ID NO:70; or the VH domain as shown in SEQ ID NO:71 paired with the VL domain as shown in SEQ ID NO:72.

14. The kit according to claim 13, wherein the one or more binding partners comprises a detectable label.

15. The kit according to claim 13, wherein the kit further comprises a tube for contacting the sample of body fluid from the subject and the TSH receptor so as to permit the TSH receptor to interact with either autoantibodies to a TSH receptor present in the sample, or the one or more binding partners.

16. The kit according to claim 13, wherein the kit further a labelled binding partner for TSH receptor autoantibodies, a competitor capable of competing with autoantibodies to a TSH receptor in the interaction thereof with a TSH receptor, or a detectable label.

17. A binding partner for a TSH receptor, the binding partner comprising an isolated monoclonal antibody or antigen binding fragment thereof that:
has a binding affinity for the TSH receptor of at least about 108 molar−1;
inhibits TSH binding, the binding partner inhibiting binding of TSH to the TSH receptor in a coated tube binding assay by 61% to 83.3% at a concentration of 2 μg/ml to 20 μg/ml wherein the TSH receptor is immobilized on a surface of the tube; and
has heavy chain variable region CDRs contained within a heavy chain variable domain having an amino acid sequence selected from the group consisting SEQ ID NOs: 67, 69, and 71.

18. A binding partner for a TSH receptor of claim 17, the binding partner comprising an isolated monoclonal antibody or antigen binding fragment thereof further comprising light chain variable region CDRs contained within a light chain variable domain having an amino acid selected from the group consisting of SEQ ID NOs 68, 70, and 72.

19. A binding partner for a TSH receptor, the binding partner comprising an isolated monoclonal antibody or antigen binding fragment thereof that:
binds to an epitope on the TSH receptor;
has a binding affinity for the TSH receptor of at least about 108 molar−1;
is positive for thyroid stimulating activity at a concentration of 20 μg/ml in a cyclic AMP thyroid cell assay, wherein the binding partner is classified as positive if the thyroid stimulating activity of the binding partner is greater than 180 percent in the cyclic AMP thyroid cell assay, said percent thyroid stimulating activity comprising 100×(ratio of cyclic AMP produced in the thyroid cell assay in the presence of said binding partner to cyclic AMP produced in the thyroid cell assay in the presence of sera pooled from healthy blood donors); and
inhibits TSH binding, the binding partner inhibiting binding of TSH to the TSH receptor in a coated tube binding assay by 61% to 83.3% at a concentration of 2 μg/ml to 20 μg/ml wherein the TSH receptor is immobilized on a surface of the tube and wherein the monoclonal antibody comprises a VH domain comprising amino acid residues 9 to 120 of SEQ ID NO:67 and a VL domain comprising amino acid residues 9 to 107 of SEQ ID NO:68; a VH domain comprising amino acid residues 9 to 120 of SEQ ID NO:69 and a VL domain comprising amino acid residues 9 to 107 of SEQ ID NO:70 or a VH domain comprising amino acid residues 9 to 120 of SEQ ID NO:71 and a VL domain comprising amino acid residues 9 to 107 of SEQ ID NO:72.

20. A binding partner for a TSH receptor, the binding partner comprising a monoclonal antibody or fragment thereof comprising a Fab domain for a TSH receptor, wherein the antibody is capable of binding to a TSH receptor so as to stimulate the TSH receptor, which comprises:
an antibody heavy chain (HC) selected from the group consisting of:
a HC comprising (SEQ ID NO:67), (SEQ ID NO:69) or (SEQ ID NO:71); and/or

an antibody light chain (LC) selected from the group consisting of:
a LC comprising (SEQ ID NO:68), (SEQ ID NO:70) or (SEQ ID NO:72).

21. The binding partner comprising a monoclonal antibody or fragment thereof of claim 20, wherein the binding partner comprising a monoclonal antibody or fragment thereof comprises at least one HC comprising (SEQ ID NO:67), (SEQ ID NO:69) or (SEQ ID NO:71) and at least one LC comprising (SEQ ID NO:68), (SEQ ID NO:70) or (SEQ ID NO:72).

22. An antibody or fragment according to claim 20, wherein the HC and LC are paired, and wherein the HC in (SEQ ID NO:67) is paired with an antibody LC in (SEQ ID NO:68) to provide an antibody binding site, comprising both these heavy and light chains for a TSH receptor, or wherein an antibody HC as shown in (SEQ ID NO:69) is paired with an antibody LC in (SEQ ID NO:70) to provide an antibody binding site comprising both these heavy and light chains for a TSH receptor, or wherein an antibody HC in (SEQ ID NO:71)is paired with an antibody LC in (SEQ ID NO:72) to provide an antibody binding site comprising both heavy and light chains for a TSH receptor.