Patent Grant
8753637
The present invention is concerned with binding partners (such as monoclonal or recombinant antibodies) for the thyrotropin receptor (TSH receptor or TSHR) and uses thereof.
Thyrotropin 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 TSH 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 serum controls the release of TSH. The formation of T3 and T4 by 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 Graves' disease (a common autoimmune disorder) TSH receptor antibodies (TRAb) are formed and these autoantibodies bind to the TSH receptor in such a way as to 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” Balliere's Clinical Endocrinology and Metabolism; Ed T F Davies 1997; 11 (3): 451-479; pub Ballière Tindall, London).
Measurements of TSH receptor antibodies are important in the diagnosis and management of Graves' disease and other thyroid disorders. Currently three types of assay are used to measure TSH receptor antibodies:
(a) competitive binding assays which measure the ability of TSH receptor antibodies to inhibit the binding of TSH to preparations of TSH receptor;
(b) bioassays which measure the ability of TSH receptor antibodies to stimulate cells expressing the TSH receptor in culture; and
(c) immunoprecipitation of TSH receptor preparations with TSH receptor antibodies.
Measurement of TSH receptor antibodies using such assays are described in references:—
It has been recognised for many years that human monoclonal antibodies to the TSH receptor derived from patients'lymphocytes would be valuable reagents for understanding the pathogenesis of Graves' disease and for developing new methods of measuring TSH receptor antibodies for example as replacements for TSH in competitive binding assays. Also, as the patient's serum TSH receptor antibodies are usually powerful thyroid stimulators (TSH agonists) stimulating human monoclonal TSH receptor antibodies would be valuable for in vivo applications when tissue containing the TSH receptor (eg thyroid tissue or thyroid cancer tissue) required stimulation. Furthermore, as some patient serum TSH receptor antibodies are powerful TSH antagonists (blocking antibodies) human monoclonal TSH receptor antibodies which are TSH antagonists would be valuable for in vivo applications when the activity of tissue containing the TSH receptor (eg thyroid tissue or thyroid cancer tissue) required inactivation or to be made unresponsive to TSH, TSH receptor antibodies or other stimulators.
It has also been recognised that one of the major advantages of human monoclonal TSH receptor antibodies over TSH in such in vitro and/or in vivo applications would be the relative ease with which antibodies can be manipulated. For example, manipulation of the TSH receptor binding region of the monoclonal antibodies so as to change their characteristics, such as affinity and biological characteristics including their degree of TSH agonist or antagonist activities. Also, monoclonal antibodies will have a much longer half life than TSH in vivo and this may have considerable advantages in certain in vivo applications. Furthermore, the half life of antibodies can be manipulated easily, for example antibody Fab fragments have a much shorter half life than intact IgG. These general properties of TSH receptor antibodies are described in the publications such as B Rees Smith, S M McLachlan, J Furmaniak; “Autoantibodies to the thyrotropin receptor”; Endocrine Reviews 1988; 9: 106-121; B Rees Smith, K J Dorrington, D S Munro; “The thyroid stimulating properties of long-acting thyroid stimulator yG-globulin subunits”; Biochimica et Biophysica Acta 1969; 192: 277-285; K J Dorrington, D S Munro; “The long acting thyroid stimulator”; Clinical Pharmacology and Therapeutics 1966; 7: 788-806.
A still further advantage of human monoclonal TSH receptor antibodies could be in their use to identify and provide new types of TSH receptor antibody binding sites. For example by the generation of antibodies to the regions of the human monoclonal TSH receptor antibodies which bind the TSH receptor. Some of the anti-idiotypic antibodies produced in this way could have potential as new ligands for assays of TSH receptor antibodies, TSH and related compounds. Also they may be effective agents in vivo for regulating the action of TSH receptor antibodies, TSH and related compounds.
Other methods of identifying and providing new types of antibody binding sites using monoclonal antibodies are well known. For example by antibody screening of phage-displayed random peptide libraries as described by J C Scott and G P Smith; “Searching for peptide ligands with an epitope library”; Science 1990; 249 (4967): 386-390 and M A Myers, J M Davies, J C Tong, J Whisstock, M Scealy, I R MacKay, M J Rowley; “Conformational epitopes on the diabetes autoantigen GAD65 identified by peptide phage display and molecular modelling”; Journal of Immunology 2000; 165: 3830-3838. Antibody screening of non-peptide compounds and libraries of non-peptide compounds can also be carried out.
New types of TSH receptor antibody binding sites identified and provided using these procedures may also be useful as new ligands in assays for TSH receptor antibodies, TSH and related compounds. Furthermore they may be effective agents in vivo for regulating the action of TSH receptor antibodies, TSH and related compounds.
In view of the potential value of human monoclonal TSH receptor antibodies there have been considerable efforts over many years to produce such antibodies (see for example B Rees Smith, S M McLachlan, J Furmaniak; “Autoantibodies to the thyrotropin receptor”; Endocrine Reviews 1988; 9: 106-121. However, to date these efforts have been unsuccessful (see for example S M McLachlan, B Rapoport; “Monoclonal, human autoantibodies to the TSH receptor—The Holy Grail and why are we looking for it”; Journal of Clinical Endocrinology and Metabolism 1996; 81: 3152-3154 and J H W van der Heijden, T W A de Bruin, K A F M Gludemans, J de Kruif, J P Banga, T Logtenberg; “Limitations of the semisynthetic library approach for obtaining human monoclonal autoantibodies to the thyrotropin receptor of Graves' disease”; Clinical and Experimental Immunology 1999; 118: 205-212).
The present provides a binding partner for TSH receptor. The binding partner comprises or is derived from
It is an object of the present invention to provide a binding partner for the TSH receptor capable of interacting with the TSH receptor in a manner comparable to the interaction of TSH receptor autoantibodies with the TSH receptor, in particular it is an object of the present invention to provide human monoclonal antibodies to the TSH receptor exhibiting a comparable interaction therewith as seen with TSH receptor antibodies present in the sera of patients with hyperthyroid Graves' disease and also to provide recombinant preparations thereof. The considerable difficulties of producing human monoclonal TSH receptor antibodies have been overcome in the invention described herein. In particular the successful production of a human monoclonal TSH receptor antibody with the characteristics of the autoantibodies found in the sera of patients with hyperthyroid Graves' disease is described. The human TSH receptor monoclonal antibody we have produced (described herein as hMAb TSHR 1) binds to the TSH receptor with high affinity and in such a way that small amounts of the antibody inhibit labelled TSH binding to the TSH receptor and small amounts act as powerful thyroid stimulators. Fab fragments of the antibody and recombinant Fab preparations are similarly effective thyroid stimulators and inhibitors of labelled TSH binding as intact IgG. Monoclonal Fab and/or intact IgG can be labelled with 125I or biotin and shown to bind to the TSH receptor. Such binding is inhibited by TSH receptor autoantibodies in patient sera.
There is provided by the present invention, therefore, a binding partner for the TSH receptor, which binding partner comprises, or is derived from, a human monoclonal or recombinant antibody, or one or more fragments thereof, reactive with the TSH receptor.
In particular, there is provided by the present invention a binding partner for the TSH receptor, which binding partner comprises, or is derived from, a human monoclonal antibody, or one or more fragments thereof, reactive with the TSH receptor.
In particular, there is provided by the present invention a binding partner for the TSH receptor, which binding partner comprises, or is derived from, a human recombinant antibody, or one or more fragments thereof, reactive with the TSH receptor.
In particular, there is provided by the present invention a human monoclonal antibody, or one or more fragments thereof, reactive with the TSH receptor.
In particular, there is provided by the present invention a human recombinant antibody, or one or more fragments thereof, reactive with the TSH receptor. Particularly, the present invention provides one or more fragments of a human recombinant antibody reactive with the TSH receptor.
A binding partner according to the present invention, and in particular, a human monoclonal or recombinant antibody reactive with the TSH receptor according to the present invention can be further characterised by its ability to inhibit TSH binding to the TSH receptor, and/or its ability to stimulate the TSH receptor, both of which have been seen to be comparable to the respective inhibitory and stimulatory properties of TSH receptor autoantibodies present in sera obtained from patients with Graves' disease.
More particularly, a binding partner according to the present invention, and in particular a human monoclonal or recombinant antibody according to the present invention, can be characterised by an inhibitory activity with respect to TSH binding to the TSH receptor, of at least about 15 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 30 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 60 units of International Standard NIBSC 90/672 per mg, or more preferably of at least about 120 units of International Standard NIBSC 90/672 per mg, or one or more fragments of such a monoclonal or recombinant antibody.
More particularly, a binding partner according to the present invention, and in particular a human monoclonal or recombinant antibody according to the present invention, can be further characterised by a stimulatory activity with respect to cAMP production by cells expressing the TSH receptor, of at least about 30 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 60 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 120 units of International Standard NIBSC 90/672 per mg, or more preferably of at least about 240 units of International Standard NIBSC 90/672 per mg, or one or more fragments of such a monoclonal or recombinant antibody.
In a preferred embodiment of the present invention, a binding partner according to the present invention, and in particular a human monoclonal or recombinant antibody according to the present invention, can be characterised by:
In the case where a binding partner according to the present invention comprises or is derived from one or more fragments of a monoclonal or recombinant antibody reactive with the TSH receptor, in particular for example one or more Fab fragments of a monoclonal or recombinant antibody reactive with the TSH receptor, it may be preferred that such a binding partner can be characterised by an inhibitory activity with respect to TSH binding to the TSH receptor, of at least about 30 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 60 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 120 units of International Standard NIBSC 90/672 per mg, or more preferably of at least about 240 units of International Standard NIBSC 90/672 per mg.
It may also be preferred in the case where a binding partner according to the present invention comprises or is derived from one or more fragments of a monoclonal or recombinant antibody reactive with the TSH receptor, in particular for example one or more Fab fragments of a monoclonal or recombinant antibody reactive with the TSH receptor, that such a binding partner can be characterised by a stimulatory activity with respect to cAMP production by cells expressing the TSH receptor, of at least about 50 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 100 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 200 units of International Standard NIBSC 90/672 per mg, or more preferably of at least about 400 units of International Standard NIBSC 90/672 per mg.
It may be still further preferred in the case where a binding partner according to the present invention comprises or is derived from one or more fragments of a monoclonal or recombinant antibody reactive with the TSH receptor, in particular for example one or more Fab fragments of a monoclonal or recombinant antibody reactive with the TSH receptor, that such a binding partner can be characterised by:
In a preferred case the present invention provides a binding partner for the TSH receptor (typically a human monoclonal antibody), which binding partner is capable of binding to the TSH receptor preferably so as to stimulate the TSH receptor and which comprises an antibody VH domain selected from the group consisting of a VH domain as shown in SEQ ID NO. 1 and a VH domain comprising one or more VH CDRs with an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.
In a first embodiment of the present invention, there is, therefore, provided a binding partner for the TSH receptor (typically a human monoclonal antibody), which binding partner is capable of binding to the TSH receptor preferably so as to stimulate the TSH receptor and which comprises an antibody VH domain as shown in SEQ ID NO. 1.
In a second embodiment of the present invention there is, therefore, provided a binding partner for the TSH receptor (typically a human monoclonal antibody), which binding partner is capable of binding to the TSH receptor preferably so as to stimulate the TSH receptor and which comprises an antibody VH domain comprising one or more VH CDRs with an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.
It will be appreciated that a binding partner according to the present invention can comprise an antibody VH domain substantially as hereinbefore described in the absence of an antibody VL domain. It is known that single immunoglobulin domains, especially VH domains, are capable of binding target antigens in a specific manner. Alternatively, a binding partner according to the present invention can comprise an antibody VH domain paired with an antibody VL domain to provide an antibody binding site comprising both VH and VL domains for a TSH receptor employing techniques well known in the art (Biochim. Biophys. Acta, 192 (1969) 277-285; Proc. Natl. Acad. Sci. USA, Vol. 89, pp 10026-10030, November 1992).
In a preferred case the present invention provides, however, a binding partner for the TSH receptor, which binding partner is capable of binding to the TSH receptor preferably so as to stimulate the TSH receptor and which comprises:
an antibody VH domain selected from the group consisting of:
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 the 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 the 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 SEQ ID NO. 1 paired with an antibody VL domain as shown in SEQ ID NO. 6 to provide an antibody binding site, comprising both these VH and VL domains for the 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 a further embodiment of the present invention there is provided a binding partner substantially as hereinbefore described for the 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:
One or more CDRs as referred to above may be taken from the hereinbefore described V1 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 hMAb TSHR 1 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, the present invention may provide 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 the TSH receptor or may have substantially no role in affecting the binding properties of the polypeptide for the TSH receptor.
The present invention, also encompasses variants, analogs, derivatives and fragments of the specific human monoclonal antibody described herein, VH domains, CDRs and polypeptides disclosed herein, which variants, analogs, derivatives and fragments retain the ability to interact with the TSH receptor (such as for example to stimulate the TSH receptor) substantially as hereinbefore described.
The terms“variants”, “analogs”, “derivatives” and“fragments” as used herein can be characterised as antibodies, antibody fragments or polypeptides which retain essentially the same biological function or activity as a human monoclonal antibody having a VH domain as shown in SEQ ID NO. 1 and a VL domain as shown in SEQ ID NO. 6 and in particular in respect of the binding properties thereof for the TSH receptor. Suitably, variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments as described herein, have a primary structural conformation of amino acids in which several or a few (such as 5 to 10, 1 to 5 or 1 to 3) amino acid residues of a human monoclonal antibody having a VH domain as shown in SEQ ID NO. 1 and a VL domain as shown in SEQ ID NO. 6 are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions which do not alter or substantially alter the biological activity or function of a human monoclonal antibody having a VH domain as shown in SEQ ID NO. 1 and a VL domain as shown in SEQ ID NO. 6. Conservative substitutions can be preferred as hereinafter described in greater detail.
More particularly, variants, analogs or derivatives of a human monoclonal antibody having a VH domain as shown in SEQ ID NO. 1 and a VL domain as shown in SEQ ID NO. 6 according to the present invention may be 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 ones in which one or more of the amino acid resides includes a substituent group or the like. Such variants, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
Most typically, variants, analogs or derivatives are those that vary from a reference human monoclonal antibody having a VH domain as shown in SEQ ID NO. 1 and a VL domain as shown in SEQ ID NO. 6 by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid 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 will be appreciated that the term fragment as used herein in particular relates to fragments of antibodies specifically as herein described and form an important aspect of the present invention.
In this way, a human monoclonal or recombinant antibody as provided by the present invention may be provided as any of the following fragments: (i) the Fab fragment consisting of VL, VH, CL and CH 1 domains; (ii) the Fd fragment consisting of the VH and CH 1 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, a human monoclonal or recombinant antibody according to the present invention may comprise a whole IgG antibody, whereby the antibody includes variable and constant regions.
The present invention also provides a further binding partner capable of binding to the TSH receptor, which can compete for binding to the TSH receptor with a binding partner for the TSH receptor (typically a human monoclonal antibody) substantially as hereinbefore described, which further binding partner does not comprise TSH. Preferably, this further binding partner may comprise a further antibody having a binding site for an epitope region of the TSH receptor, and which can compete for binding to the TSH receptor with a binding partner for the TSH receptor (typically a human monoclonal antibody) substantially as hereinbefore described. A suitable such further binding partner can comprise a mouse monoclonal antibody, which can preferably be produced according to techniques substantially as described in the Examples, employing immunisation of mice with TSH receptor by techniques known in the art.
The present invention may also provide a further binding partner capable of binding to the TSH receptor, which can comprise, or is derived from, a human monoclonal or recombinant antibody, or one or more fragments thereof, reactive with the TSH receptor. In particular this further binding partner may comprise a further antibody having a binding site for an epitope region of the TSH receptor, which further antibody is capable of binding to the TSH receptor, and can compete for binding to the TSH receptor with a binding partner for the TSH receptor (typically a human monoclonal antibody) substantially as hereinbefore described. Suitably such a further binding partner can be derived from a specific binding partner as described herein, hMAb TSHR 1, by suitable mutagenesis techniques, such as spot mutations or the like, so as to obtain a further binding partner for the TSH receptor that can compete with a binding partner substantially as herein described (such as hMAb TSHR 1) for interaction with the TSH receptor.
Preferably a further binding partner for the TSH receptor can comprise a monoclonal or recombinant antibody and can be characterised by an inhibitory activity with respect to TSH binding to the TSH receptor, of at least about 15 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 30 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 60 units of International Standard NIBSC 90/672 per mg, or more preferably of at least about 120 units of International Standard NIBSC 90/672 per mg, or one or more fragments of the antibody. It may also be preferred that such a further binding partner according to the present invention, can be characterised by a stimulatory activity with respect to cAMP production by cells expressing the TSH receptor, of at least about 30 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 60 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 120 units of International Standard NIBSC 90/672 per mg, or more preferably of at least about 240 units of International Standard NIBSC 90/672 per mg, or one or more fragments of the antibody.
It may also be even more preferred that such a further binding partner of the present invention, can be characterised by:
A preferred mouse monoclonal antibody providing a further binding partner according to the present invention comprises 9D33 prepared further to the Examples and having amino acid and polynucleotide sequences as illustrated by
A further binding partner as provided by the present invention can also be characterised as comprising an antibody VH domain comprising one or more VH CDRs with an amino acid sequence selected from SEQ ID NO. 20, SEQ ID NO. 21 and SEQ ID NO. 22.
It will be appreciated that a further binding partner according to the present invention can comprise an antibody VH domain substantially as hereinbefore described in the absence of an antibody VL domain. It is known that single immunoglobulin domains, especially VH domains, are capable of binding target antigens in a specific manner. Alternatively, a further binding partner according to the present invention can comprise an antibody VH domain paired with an antibody VL domain to provide an antibody binding site comprising both VH and VL domains for a TSH receptor employing techniques well known in the art (Biochim. Biophys. Acta, 192 (1969) 277-285; Proc. Natl. Acad. Sci. USA, Vol. 89, pp 10026-10030, November 1992).
In a preferred case the present invention provides, however, a further binding partner for the TSH receptor, which further binding partner comprises:
an antibody VH domain selected from the group consisting of:
It may be preferred according to the present invention that a further 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 the 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 further 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 further 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 further 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 the TSH receptor.
Preferred embodiments according to the present invention can thus include a further binding partner substantially as hereinbefore described comprising an antibody VH domain as shown in SEQ ID NO. 19 paired with an antibody VL domain as shown in SEQ ID NO. 24 to provide an antibody binding site, comprising both these VH and VL domains for the 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 a further embodiment of the present invention there is provided a further binding partner substantially as hereinbefore described for the TSH receptor, which further binding partner is capable of binding to the TSH receptor so as to inhibit stimulation of the TSH receptor and which can comprise:
an antibody VH domain comprising:
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 9D33 specifically disclosed herein, such antibodies thereby incorporating the one or more CDRs and being capable of binding to the TSH receptor. Alternatively, the present invention may provide a polypeptide capable of binding to the TSH receptor 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 the TSH receptor or may have substantially no role in affecting the binding properties of the polypeptide for the TSH receptor.
It will be appreciated that the term fragment as used herein in particular relates to fragments of antibodies specifically as herein described and form an important aspect of the present invention. In this way, a further binding partner according to the present invention may be provided as any of the following fragments: (i) the Fab fragment consisting of VL, VH, CL and CH 1 domains; (ii) the Fd fragment consisting of the VH and CH 1 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, a mouse monoclonal or recombinant antibody according to the present invention, such as 9D33, may comprise a whole IgG antibody, whereby the antibody includes variable and constant regions.
There is also provided by the present invention a polynucleotide comprising:
There is also provided by the present invention a polynucleotide comprising:
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.
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 for the TSH receptor of the invention by recombinant techniques. Host cells can be genetically engineered to incorporate polynucleotides and express binding partners for the TSH receptor of the present invention.
The amino acid sequences of hMAb TSHR 1, a human monoclonal antibody according to the present invention, and nucleotide sequences coding therefor, the amino acid sequences of 9D33, a mouse monoclonal antibody which represents a further binding partner according to the present invention, and nucleotide sequences coding therefor, are shown in the Sequence listings as herein after described and can be assigned as follows.
For hMAb TSHR 1:
For 9D33:
The above sequences for hMab TSHR1 can also be seen by reference to FIGS. 4,5, 6 and 7, wherein:
It will be appreciated from the above that for the VH chain of hMab TSHR1 the nucleotide sequences of the CDRI, CDRII and CDRIII regions as shown in
Analysis of the crystal structure of hMAb TSHRI Fab (determined by techniques known in the art) enabled refinement of the HC and LC nucleotide sequences determined using PCR primers which are degenerate. In particular, a HC sequencing artefact for nucleotides 115-120 was identified. Sequencing indicated cacgtg (transcribed to amino acids H is Val), whereas the crystal structure more reliably indicated amino acids Gln Leu (corresponding bases being cagctg), with the refined sequences being shown in the accompanying Figures and Sequence listings. Crystal structure analysis also enabled refinement of the HC and LC derived amino acid sequences particularly in the degenerate PCR primer region. In the case of the LC aa 2 was found to be Pro by RT-PCR but was Thr from the crystal structure. In the case of the HC aa 2 was found to be Met by RT-PCR but was Val from the crystal structure. Again, these refined sequences are shown in the accompanying Figures and Sequence listings.
The above sequences for 9D33 can also be seen by reference to
It will be appreciated from the above that for the VH chain of 9D33 the nucleotide sequences of the CDRI, CDRII and CDRIII regions as shown in
The present invention also provides a process of providing a human monoclonal antibody to the TSH receptor substantially as hereinbefore described, which process comprises:
Alternatively, a process of providing a human monoclonal antibody to the TSH receptor substantially as hereinbefore described can be defined as a process which comprises:
Preferably a process according to the present invention comprises isolating lymphocytes from peripheral blood, thyroid tissue, spleen tissue, lymph nodes or bone marrow, most typically from peripheral blood. Typically, the source of lymphocytes for use in a method according to the present invention can be further characterised as being obtained from a subject having serum TSH receptor antibody levels of greater than about 0.1 units of NIBSC 90/672 per mL with respect to inhibition of TSH binding to the TSH receptor, or more typically greater than about 0.2 units of NIBSC 90/672 per mL with respect to inhibition of TSH binding to the TSH receptor, or more typically greater than about 0.3 units of NIBSC 90/672 per mL with respect to inhibition of TSH binding to the TSH receptor and preferably being in the range of about 0.3 to 0.5 units of NIBSC 90/672 per mL or greater with respect to inhibition of TSH binding to the TSH receptor. Alternatively, or additionally, the source of lymphocytes for use in a method according to the present invention can typically be further characterised as being obtained from a subject having serum TSH receptor antibody levels of greater than about 0.2 units of NIBSC 90/672 per mL with respect to stimulatory activity of cAMP production by cells expressing the TSH receptor, or more typically greater than about 0.5 units of NIBSC 90/672 per mL with respect to stimulatory activity of cAMP production by cells expressing the TSH receptor and preferably being in the range of about 0.5 to 1.0 units of NIBSC 90/672 per mL or greater with respect to stimulatory activity of cAMP production by cells expressing the TSH receptor. It will be appreciated from the above that the immune response to the TSH receptor of a subject from which lymphocytes are isolated should preferably be in a highly active phase.
Preferably a process according to the present invention comprises infecting the isolated lymphocytes with Epstein Barr virus, and suitably the thus immortalised lymphocytes are fused with a mouse/human cell line. Suitably a process according to the present invention further comprises screening the resulting clones for TSH receptor antibodies, for example by inhibition of 125I TSH binding to the TSH receptor in an assay system which has a sensitivity of at least about 1 unit/L of NIBSC 90/672.
The present invention further provides a process of preparing a human recombinant antibody, or one or more fragments thereof, to the TSH receptor, which process comprises cloning and expression of a human monoclonal antibody to the TSH receptor as provided by the present invention by a process substantially as hereinbefore described, or one or more fragments derived therefrom.
The present invention further provides a human monoclonal or recombinant antibody to the TSH receptor obtained by a process substantially as described above. Preferably such an obtained human monoclonal or recombinant antibody to the TSH receptor according to the present invention, can be characterised by an inhibitory activity with respect to TSH binding to the TSH receptor, of at least about 15 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 30 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 60 units of International Standard NIBSC 90/672 per mg, or more preferably of at least about 120 units of International Standard NIBSC 90/672 per mg, or one or more fragments of such a human monoclonal or recombinant antibody.
More particularly, it may be preferred that such a human monoclonal or recombinant antibody according to the present invention, can be further characterised by a stimulatory activity with respect to cAMP production by cells expressing the TSH receptor, of at least about 30 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 60 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 120 units of International Standard NIBSC 90/672 per mg, or more preferably of at least about 240 units of International Standard NIBSC 90/672 per mg, or one or more fragments of such a human monoclonal or recombinant antibody.
In a preferred embodiment of the present invention, such a human monoclonal or recombinant antibody according to the present invention, can be characterised by:
It may also be preferred that one or more fragments of a thus obtained human monoclonal or recombinant antibody according to the present invention, in particular for example one or more Fab fragments thereof, can be characterised by an inhibitory activity with respect to TSH binding to the TSH receptor, of at least about 30 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 60 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 120 units of International Standard NIBSC 90/672 per mg, or more preferably of at least about 240 units of International Standard NIBSC 90/672 per mg. It may also be preferred that such one or more fragments can be characterised by a stimulatory activity with respect to cAMP production by cells expressing the TSH receptor, of at least about 50 units of International Standard NIBSC 90/672 per mg, or more preferably of at least about 100 units of International Standard NIBSC 90/672 per mg, or more preferably of at least about 200 units of International Standard NIBSC 90/672 per mg, or more preferably of at least about 400 units of International Standard NIBSC 90/672 per mg.
More preferably, such one or more Fab fragments can be characterised by:
A process substantially as described above may further comprise a further process stage whereby the obtained human monoclonal or recombinant antibody is subjected to suitable further processing techniques (such as suitable mutagenesis techniques, such as spot mutations or the like), so as to obtain a further binding partner for the TSH receptor that can compete with a binding partner substantially as herein described (such as hMAb TSHR 1) for interaction with the TSH receptor. Such further processing techniques are well known to one of ordinary skill in the art. The present invention further provides a further binding partner to the TSH receptor obtained by such further processing techniques.
Preferably such a further binding partner for the TSH receptor can comprise a monoclonal or recombinant antibody and can be characterised by an inhibitory activity with respect to TSH binding to the TSH receptor, of at least about 15 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 30 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 60 units of International Standard NIBSC 90/672 per mg, or more preferably of at least about 120 units of International Standard NIBSC 90/672 per mg, or one or more fragments thereof. It may also be preferred that such a further binding partner according to the present invention, can be characterised by a stimulatory activity with respect to cAMP production by cells expressing the TSH receptor, of at least about 30 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 60 units of International Standard NIBSC 90/672 per mg, more preferably of at least about 120 units of International Standard NIBSC 90/672 per mg, or more preferably of at least about 240 units of International Standard NIBSC 90/672 per mg, or one or more fragments thereof.
It may also be even more preferred that such a further binding partner of the present invention, can be characterised by:
A binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) according to the present invention may have diagnostic and therapeutic applications.
Accordingly, a binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) according to the present invention can be employed in screening methods for detecting autoantibodies to the TSH receptor in patient sera and also in diagnostic methods. In this way, a binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) according to the present invention can be employed in place of, or in addition to, competitors hitherto described for use in screening methods for detecting autoantibodies to the TSH receptor and also in diagnostic methods. Similarly, a binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) according to the present invention can be employed in place of, or in addition to, competitors hitherto described for use in kits for use in detecting autoantibodies to the TSH receptor.
The present invention also provides, therefore, a method of screening for autoantibodies to the 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 method according to the present invention for the detection of autoantibodies as described above is particularly advantageous in terms of the level of sensitivity that can be achieved by use thereof. This can be further illustrated by reference to the Examples and Figures, where
There is further provided by the present invention, therefore, a method of screening for autoantibodies to the 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 the TSH receptor, said method comprising:
The above sensitivity can also be achieved in an assay method or kit according to the present invention by the use of a human or non-human polyclonal antibody to the TSH receptor, TSH or one or more variants, analogs, derivatives or fragments thereof, or a binding partner for the TSH receptor which has an affinity for the TSH receptor of 1010 molar−1 or greater, which generally exhibit a sufficient affinity for the TSH receptor so that a method or kit of the defined sensitivity is provided. The preparation of such polyclonal antibodies, TSH or one or more variants, analogs, derivatives or fragments thereof, is well known in the art. For example, superactive analogs of TSH are described in Nature, Biotechnology, Volume 14, October 1995, pages 1257-1263, although this article does not disclose the use of such superactive TSH in a method or kit as is now provided by the present invention.
There is further provided by the present invention, therefore, a method of screening for autoantibodies to the 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 the TSH receptor, said method comprising:
There is also provided by the present invention a method of screening for autoantibodies to the 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 the TSH receptor, said method comprising:
There is also still further provided a method of screening for autoantibodies to the 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 the TSH receptor, said method comprising:
There is also provided by the present invention use of a binding partner or further binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) according to the present invention, for detecting autoantibodies to the 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 the TSH receptor, wherein the interaction of said binding partner or further binding partner with the TSH receptor is such that an autoantibody titer in said sample essentially corresponding to 0.4 U/L of International Standard NIBSC 90/672 is detectable.
There is also provided use of a human or non-human polyclonal antibody to the TSH receptor, for detecting autoantibodies to the 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 the TSH receptor, wherein the interaction of said polyclonal antibody with the TSH receptor is such that an autoantibody titer in said sample essentially corresponding to 0.4 U/L of International Standard NIBSC 90/672 is detectable.
There is also provided use of TSH or one or more variants, analogs, derivatives or fragments thereof, for detecting autoantibodies to the 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 the TSH receptor, wherein the interaction of said TSH or one or more variants, analogs, derivatives or fragments thereof with the TSH receptor is such that an autoantibody titer in said sample essentially corresponding to 0.4 U/L of International Standard NIBSC 90/672 is detectable.
There is still further provided use of a binding partner for the TSH receptor which has an affinity for the TSH receptor of 1010 molar−1 or greater, for detecting autoantibodies to the 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 the TSH receptor, wherein the interaction of said binding partner with the TSH receptor is such that an autoantibody titer in said sample essentially corresponding to 0.4 U/L of International Standard NIBSC 90/672 is detectable.
It will be appreciated that binding molecules of the one or more binding pairs can be antigen-antibody (for example, [TSH receptor or epitope]-[monoclonal or recombinant TSH receptor antibody]), anti-idiotypic antibody-monoclonal or recombinant TSH receptor antibody or novel TSH receptor antibody binding member-monoclonal or recombinant TSH receptor antibody. Preferably, the binding molecules of the binding pairs are antigen-antibody, namely, [TSH receptor or one or more epitopes thereof]-[monoclonal or recombinant TSH receptor antibody], where the epitopes may be“free standing” or present in a larger scaffold polypeptide or the like.
Preferably, the present invention provides a method of screening for autoantibodies to the 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:
In certain embodiments, a method according to the present invention may also employ one or more competitors that compete in the interaction of a polyclonal antibody, TSH or one or more variants, analogs, derivatives or fragments thereof, or a binding partner or further binding partner for the TSH receptor substantially as described above in the specific embodiments of methods as provided by the present invention and the second molecule of the binding pair, or the TSH receptor, or the one or more epitopes thereof or the polypeptide. Such competitors may comprise TSH, or one or more monoclonals reactive with the TSH receptor, such as mouse monoclonals reactive with the TSH receptor.
Preferably, a method according to the present invention as referred to above, further comprises providing labelling means for a binding partner or further binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) according to the present invention and where appropriate one or more competitors as described above, suitable labelling means including enzymic labels, isotopic labels, chemiluminescent labels, fluorescent labels, dyes and the like.
The present invention also provides, a kit for screening for autoantibodies to the 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:
The present invention also provides a kit for screening for autoantibodies to the 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 the TSH receptor, said kit comprising:
There is also provided a kit for screening for autoantibodies to the 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 the TSH receptor, said kit comprising:
There is also provided a kit for screening for autoantibodies to the 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 the TSH receptor, said kit comprising:
There is also provided a kit for screening for autoantibodies to the 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 the TSH receptor, said kit comprising:
It will be appreciated that binding molecules of the one or more binding pairs can be antigen-antibody (for example, [TSH receptor or epitope]-[monoclonal or recombinant TSH receptor antibody]), anti-idiotypic antibody-monoclonal or recombinant TSH receptor antibody or novel TSH receptor antibody binding member-monoclonal or recombinant TSH receptor antibody. Preferably, the binding molecules of the binding pairs are antigen-antibody, namely, [TSH receptor or one or more epitopes thereof]-[monoclonal or recombinant TSH receptor antibody], where the epitopes may be“free standing” or present in a larger scaffold polypeptide or the like.
The present invention preferably provides a kit for screening for autoantibodies to the 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 the TSH receptor, said kit comprising:
In certain embodiments, a kit according to the present invention may further comprise one or more competitors that compete in the interaction of a polyclonal antibody, TSH or one or more variants, analogs, derivatives or fragments thereof, or a binding partner or further binding partner for the TSH receptor, as respectively defined above, and the second molecule of the binding pair, or the TSH receptor, or the one or more epitopes thereof or the polypeptide. Such competitors may comprise TSH, or one or more monoclonals reactive with the TSH receptor, such as mouse monoclonals reactive with the TSH receptor.
Suitably, a kit as referred to above further comprises labelling means for a binding partner or further binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) according to the present invention and where appropriate one or more competitors as described above, suitable labelling means being substantially as hereinbefore described.
In the presence of autoantibodies to the TSH receptor, binding of the TSH receptor to a binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) in a method or kit as described above will be decreased.
A binding partner or further binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) according to the present invention can also be employed in assay methods and kits substantially as described above for TSH and related ligands.
The present invention also provides, therefore, a method of assaying TSH and related ligands, said method comprising:
The present invention also provides a kit for assaying TSH or related ligands, said kit comprising:
The present invention also further provides a method of identifying a further binding partner for the TSH receptor, which further binding partner is capable of binding to the TSH receptor and which competes for binding to the TSH receptor with a binding partner for the TSH receptor substantially as hereinbefore described, which further binding partner does not comprise TSH, which method comprises:
The present invention also provides a kit for identifying a further binding partner for the TSH receptor, which further binding partner is capable of binding to the TSH receptor and which competes for binding to the TSH receptor with a binding partner for the TSH receptor substantially as hereinbefore described, which further binding partner does not comprise TSH, which kit comprises:
A further application of a binding partner or further binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) according to the present invention is its use to identify and provide new types of TSH receptor antibody binding sites. There is further provided by the present invention, therefore, a process of identifying one or more epitope regions of the TSH receptor, which process comprises contacting a binding partner or further binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) substantially as hereinbefore described with a full length TSH receptor, or one or more fragments thereof, so as to allow interaction of said binding partner or further binding partner for the TSH receptor with said full length TSH receptor, or said one or more fragments thereof, and identifying the amino acids of said full length TSH receptor, or said one or more fragments thereof, with which said binding partner or further binding partner interacts. Suitably, interaction of the binding partner or further binding partner with selected fragments of the TSH receptor and the full length TSH receptor, is analysed, so as to identify the amino acids of the TSH receptor with which the binding partner interacts.
Furthermore, the present invention allows for generation of antibodies to the regions of a monoclonal TSH receptor antibody according to the present invention which bind the TSH receptor. Such anti-idiotypic antibodies produced in this way could have potential as new ligands for assays of TSH receptor autoantibodies, TSH and related compounds. Also they may be effective agents in vivo for regulating the action of TSH receptor autoantibodies, TSH and related compounds. The present invention further provides, therefore, one or more anti-idiotypic antibodies generated to binding regions of a binding partner or further binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) substantially as hereinbefore described, and the preparation thereof is further described by the Examples.
Other methods of identifying and providing new types of antibody binding sites using monoclonal antibodies are well known. For example by antibody screening of phage-displayed random peptide libraries as described by J C Scott and G P Smith; “Searching for peptide ligands with an epitope library”; Science 1990; 249 (4967): 386-390 and MA Myers, J M Davies, J C Tong, J Whisstock, M Scealy, I R MacKay, M J Rowley; “Conformational epitopes on the diabetes autoantigen GAD65 identified by peptide phage display and molecular modelling”; Journal of Immunology 2000; 165: 3830-3838. Antibody screening of non-peptide compounds and libraries of non-peptide compounds can also be carried out.
New types of TSH receptor antibody binding sites identified and provided using these procedures may also be useful as new ligands in assays for TSH receptor autoantibodies, TSH and related compounds. Furthermore they may be effective agents in vivo for regulating the action of TSH receptor autoantibodies, TSH and related compounds.
A binding partner for the TSH receptor or further binding partner (typically a human monoclonal or recombinant antibody) according to the present invention 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 binding partner or further binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) substantially as hereinbefore described, pharmaceutical compositions comprising a binding partner or further binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) substantially as hereinbefore described (together with one or more pharmaceutically acceptable carriers, diluents or excipients therefor), and use of a binding partner or further binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) substantially as hereinbefore described in the manufacture of a medicament or composition.
A binding partner for the TSH receptor, in particular a human monoclonal antibody to the TSH receptor derived from patients' lymphocytes according to the present invention, is a valuable reagent for understanding the pathogenesis of Graves' disease and for developing new methods of measuring TSH receptor autoantibodies, for example as replacements for TSH in competitive binding assays substantially as hereinbefore described. Also, a stimulating binding partner according to the present invention has in vivo applications when tissue containing the TSH receptor (eg thyroid tissue or thyroid cancer tissue) requires stimulation. The present invention provides, therefore, a medicament or composition for use in stimulating thyroid tissue, and/or tissue containing the TSH receptor. In particular, a stimulating binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) according to the present invention can be employed in oncology, and in particular for use in the diagnosis, management and treatment of thyroid cancer.
Alternatively, a binding partner or further binding partner for the TSH receptor according to the present invention can be a powerful TSH or autoantibody antagonist (blocking antibody) and such a blocking TSH receptor antibody according to the present invention is valuable for in vivo applications when the activity of tissue containing the TSH receptor (eg thyroid tissue or thyroid cancer tissue) requires inactivation or to be made unresponsive to TSH, TSH receptor autoantibodies or other stimulators.
There is also provided in combination, a binding partner or further binding partner for the TSH receptor substantially as hereinbefore described, together with one or more further agents capable of inactivating or rendering unresponsive, tissue containing a TSH receptor, to TSH, TSH receptor autoantibodies or other stimulators. Typically, the one or more further agents act independently of the TSH receptor.
A particular therapeutic application where TSH receptor autoantibody binding requires inactivation or inhibition is in the treatment of disease of the retro orbital tissues of the eye associated with autoimmunity to the TSH receptor, and the use of a blocking antibody which interacts with the TSH receptor, such as 9D33, so as to inhibit TSH receptor autoantibody binding, thus has important therapeutic utility in the treatment of such disease. Treatment of autoimmune disease which requires inhibition of TSH receptor autoantibody binding, such as the above discussed disease of the retro orbital tissues of the eye associated with autoimmunity to the TSH receptor, may alternatively employ an anti-idiotypic antibody to a binding partner or further binding partner as provided by the present invention, and such anti-idiotypic antibodies form a further aspect of the present invention as described herein and further preparatory details thereof are provided by the Examples.
More specifically, therefore, the present invention provides use in the treatment of disease of the retro orbital tissues of the eye associated with autoimmunity to the TSH receptor, of a further binding partner to the TSH receptor, which further binding partner substantially inhibits binding to the TSH receptor of a binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) substantially as hereinbefore described. The present invention further provides use in the manufacture of a medicament for the treatment of disease of the retro orbital tissues of the eye associated with activation and/or stimulation of the TSH receptor, of a further binding partner to the TSH receptor, which further binding partner substantially inhibits binding to the TSH receptor of a binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) substantially as hereinbefore described. There is also provided a method of treating disease of the retro orbital tissues of the eye associated with autoimmunity to the TSH receptor, which method comprises administration to a patient suffering from or susceptible to such disease a therapeutically effective amount of a further binding partner to the TSH receptor, which further binding partner substantially inhibits binding to the TSH receptor of a binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) substantially as hereinbefore described. A further binding partner for use in these embodiments of the present invention preferably comprises a blocking antibody which can substantially inhibit binding of a binding partner as provided by the present invention, and as such TSH receptor autoantibody binding, to the TSH receptor, and a preferred such antibody can comprise 9D33 as described herein.
The present invention also provides use of an anti-idiotypic antibody generated to a binding region of a binding partner or further binding partner according to the present invention, in the treatment of disease of the retro orbital tissues of the eye associated with autoimmunity to the TSH receptor. The present invention further provides use of an anti-idiotypic antibody generated to a binding region of a binding partner or further binding partner according to the present invention, in the manufacture of a medicament for the treatment of disease of the retro orbital tissues of the eye associated with activation and/or stimulation of the TSH receptor. There is also provided a method of treating disease of the retro orbital tissues of the eye associated with autoimmunity to the TSH receptor, which method comprises administration to a patient suffering from or susceptible to such disease a therapeutically effective amount of an anti-idiotypic antibody generated to a binding region of a binding partner or further binding partner according to the present invention.
One of the major advantages of a monoclonal antibody as provided by the present invention over TSH in such in vitro and/or in vivo applications is the relative ease with which such antibodies can be manipulated. For example, manipulation of the TSH receptor binding region of a monoclonal antibody according to the present invention so as to change the characteristics thereof, such as affinity and biological characteristics, including the degree of TSH agonist or antagonist activities. Also monoclonal antibodies according to the present invention have a much longer half life than TSH in vivo and this may have considerable advantages in in vivo applications. Furthermore, the half life of the antibodies can be manipulated, for example antibody Fab fragments have a much shorter half life than intact IgG.
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 or further binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) 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 or further binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) substantially as hereinbefore described.
The present invention also provides in combination, a binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) 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.
A binding partner for the TSH receptor or further binding partner (typically a human monoclonal or recombinant antibody) according to the present invention can also be employed as a replacement source for patient serum required to contain TSH receptor antibody or antibodies for use in commercial kits. Furthermore, a binding partner or further binding partner for the TSH receptor (typically a human monoclonal or recombinant antibody) can be provided according to the present invention in a preparation required to comprise a defined concentration of TSH receptor antibody or antibodies, and in this way there can be provided a preparation with a defined activity, such as stimulatory activity, with respect to the TSH receptor. Optionally, such a preparation may further comprise one or more further human monoclonal antibodies, such as monoclonal antibodies to GAD, TPO or the like.
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 FOR A TSH RECEPTOR, describes a molecule having a binding specificity for the TSH receptor. A binding partner as described herein may be naturally derived or wholly or partially synthetically produced. Such a binding partner has a domain or region which specifically binds to and is therefore complementary to one or more epitope regions of the TSH receptor. In particular, a binding partner as described herein can be a monoclonal or recombinant antibody to the TSH receptor, and more particularly can be a human monoclonal or recombinant antibody to the TSH receptor.
C DOMAIN denotes a region of relatively constant amino acid sequence in antibody molecules.
CDR denotes complementarity 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.
NIBSC 90/672 is, an International Standard for thyroid stimulating antibody. The International Standard for thyroid stimulating activity consists of a batch of ampoules containing freeze dried plasma proteins from a single human patient with high TSH receptor autoantibodies. The preparation has been evaluated in an international collaborative study and shown to possess both thyroid stimulating and thyroid receptor binding activity. At the 46th meeting in 1995, the Expert Committee on Biological Standardization of WHO established the preparation coded 90/672 as the International Standard for thyroid stimulating antibody. Each ampoule contains freeze-dried residue of 1.0 ml of a solution containing 0.02M phosphate buffer, dialysed human plasma proteins and 0.1 International Units (100 milli-International Units) per ampoule by definition.
STIMULATION OF A TSH RECEPTOR by a human monoclonal antibody as described herein denotes the ability thereof 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 TSH, or TSH receptor autoantibodies, to the TSH receptor and in this way a human monoclonal antibody as described herein essentially provides the same or similar binding responses as seen with 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.
Lymphocyte Isolation and Clonina of Human Monoclonal TSH Receptor Autoantibodies
Blood was obtained from a patient with Graves' disease and Type 1 diabetes mellitus who had high levels of serum autoantibodies to the TSH receptor (TRAb). Ethical Committee approval was obtained for the studies. Peripheral blood lymphocytes were isolated on Ficoll-Paque (Amersham Biosciences; Chalfont St Giles, HPS 4SP, UK) from a 20 mL blood sample and then infected with Epstein Barr virus (EBV) (European Collection of Cell Cultures-ECACC; Porton Down, SP4 OJG, UK) and cultured on mouse macrophage feeder layers as described before (N Hayakawa, L D K E Premawardhana, M Powell, M Masuda, C Arnold, J Sanders, M Evans, S Chen, J C Jaume, S Baekkeskov, B Rees Smith, J Furmaniak; “Isolation and characterization of human monoclonal autoantibodies to glutamic acid decarboxylase”; Autoimmunity 2002; 35: 343-355). EBV immortalised B lymphocytes were then fused with the mouse/human hybrid cell line K6H6/B5 (W L Carroll, K Thilemans, J Dilley, R Levy; “Mouse x human heterohybridomas as fusion partners with human B cell tumors”; Journal of Immunological Methods 1986; 89: 61-72) and cloned two times by limiting dilution at 5 cells/well and a final time at ½ cell/well to obtain a single colony (B J Bolton, N K Spurr. “B-lymphocytes” In: R I Freshney, M G Freshney (eds). Culture of immortalized cells. Wiley-Liss, New York 1996; 283-297). The original wells and subsequent clones were screened for TSH receptor autoantibody by inhibition of 125I-TSH binding to solubilised TSH receptor (see below). The single clones producing TSH receptor autoantibodies were grown up in tissue culture flasks.
Production, Purification and Labelling of Monoclonal TSH Receptor Antibody Preparations
Mouse TSH receptor MAbs were produced as described before (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: 1051-1059) and were also prepared from mice immunised with full length TSHR cDNA cloned in pcDNA3.1 (U A Hasan, A M Abai, D R Harper, B W Wren, W J W Morrow; “Nucleic acid immunization: Concepts and techniques associated with third generation vaccines”; Journal of Immunological Methods 1999; 229: 1-22).
IgGs were purified from tissue culture supernatants using affinity chromatography on Prosep A (Millipore UK Ltd.; Watford, WD18 SYH, UK) according to the manufacturer's instructions and purity assessed by SDS-polyacrylamide gel electrophoresis (PAGE).
Human heavy chain isotype was determined using a radial diffusion assay (The Binding Site; Birmingham, B29 6AT, UK). Human light chain isotype was determined using Western blotting with anti-human kappa chain and anti human lambda chain specific mouse monoclonal antibodies (Sigma-Aldrich Company Ltd; Gillingham, SP8 4XT, UK).
The purified IgG preparations were treated with mercuripapain (Sigma-Aldrich) at an enzyme/protein ratio of between 1:10 and 1:100 (depending on the particular monoclonal antibody) and passed through a Prosep A column to remove any intact IgG or Fc fragment from the Fab preparation (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). Intact IgG was undetectable by SDS-PAGE in the Fab preparations. IgG and Fab preparations of the monoclonal antibodies were labelled with 125I as described previously (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). IgG preparations were labelled with biotin hydrazide (Pierce Rockford Ill. 61105 USA) according to the manufacturers instructions. Crystals of Fab fragments of the human monoclonal TSH receptor autoantibody were obtained and their crystal structure determined using standard techniques.
Patients
Sera from patients with Graves' disease of different disease duration were studied. The patients' sera studied showed inhibition of 125I-labelled TSH binding to the TSH receptor (see below). In addition, sera from 2 patients with Addison's disease (A1 and A2) and high levels of autoantibodies to 21-OH (113 and 1970 units per mL, RSR kit) and sera from 2 patients with type 1 diabetes mellitus (D1 and D2) with high levels of GAD65 (3700 and 37.5 units per mL; RSR kit) were studied. Informed consent for the study was obtained from the patients. Sera from healthy blood donors (purchased from Golden West Biologicals, Vista, Calif. 92083, USA) were also studied. TRAb first international standard preparation (90/672) was obtained from the National Institute for Biological Standards and Control (NIBSC; Potters Bar, EN6 3QH, UK).
Inhibition of 125I-TSH and 125I-Mouse TSHR MAb Binding to the TSH Receptor
Binding inhibition assays were carried out using TSH receptor coated tubes as described previously (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-labeled TSH receptor”; Journal of Clinical Endocrinology and Metabolism 1999; 84: 3797-3802) (reagents from RSR Ltd). Briefly, 100 μL of sample (tissue culture supernatant, purified IgG or Fab fragment, patient serum or NIBSC 90/672 standards) were incubated in TSH receptor coated tubes at room temperature for 2 hours with gentle shaking. After aspiration, the tubes were washed twice with 1 mL of assay buffer (50 mmol/L NaCl, 10 mmol/L Tris-HCl pH 7.8, 0.1% Triton X-100) before addition of 100 μL of 125I-TSH or 125I-MAb (5×104 cpm) and incubation at room temperature for 1 hour with shaking. The tubes were then washed twice with 1 mL of assay buffer, aspirated and counted in a gamma counter.
Inhibition of Binding was Calculated as:—
Control materials used were culture medium, a pool of healthy blood donor sera or as otherwise indicated.
Analysis of Thyroid Stimulating Activities
The ability of monoclonal autoantibody preparations and patient sera to stimulate the production of cyclic AMP (or cAMP) in CHO cells expressing hTSH receptor (approximately 50,000. receptors per cell) (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) were carried out according to the method of R Latif, P Graves, T F Davies; “Oligomerization of the human thyrotropin receptor”; Journal of Biological Chemistry 2001; 276: 45217-45224. Briefly, CHO cells were seeded into 96 well plates (30,000 cells per well) and incubated for 24 hours in DMEM (Invitrogen Ltd; Paisley PA4 9RF, UK) containing 10% fetal calf serum. Culture was then continued in DMEM without fetal calf serum for a further 24 hours. The DMEM was then removed and test TSH, IgG, Fab and serum (100 μl diluted in NaCl free Hank's Buffered Salts solution containing Ig/L glucose, 20 mmol/L Hepes, 222 mmol/L sucrose, 15 g/L bovine serum albumin (BSA) and 0.5 mmol/L 3 isobutyl-1-methyl xanthine pH 7.4) added and incubated for 1 hour at 37° C. After removal of the test solutions, cells were lysed and assayed for cyclic AMP using a Biotrak enzyme immunoassay system from Amersham Biosciences; Chalfont St Giles, HPS 4SP, UK. In some experiments, the ability of patient sera and mouse monoclonal antibodies to the TSHR to inhibit the stimulating activity of TSH or hMAb TSHR1 was assessed. This was carried out by comparing (a) the stimulatory effects of TSH or hMAb TSHR1 alone with (b) the stimulatory effects of TSH or hMAb TSHR1 in the presence of patient sera or mouse monoclonal antibody.
Variable Region Gene Analysis
Total RNA was prepared from 1×107 cells of a TSH receptor autoantibody producing clone using the acid phenol guanidine method (P Chomczynski, N Sacchi; “Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction”; Analytical Biochemistry 1987; 162: 156-159) and mRNA prepared using oligo dT magnetic beads (Dynal Biotech Ltd; Win-al, CH62 3QL, UK). RT-PCR reactions were performed using reagents from Invitrogen Ltd; Paisley PA4 9RF, UK.
Sense strand oligonucleotide primers were designed using the sequences recommended by the Medical Research Council's V-base database (www.mrc-cpe.cam.ac.uk). Antisense primers specific for human IgG1 heavy chain and lambda light chain were based on constant region encoding DNA sequences. Both sense and antisense primers included additional 5′ restriction endonuclease site sequences to facilitate cloning of PCR products. IgG1 heavy chain and lambda light chain RT-PCR reactions were performed using the complete panel of appropriate primers. All primers were synthesized by Invitrogen Ltd. The RT reaction took place at 50° C. for 10 minutes followed immediately by 40 cycles of PCR (15 sec 94° C., 30 sec 55° C., 30 sec 72° C.). RT-PCR products were cloned into pUC18 and DNA prepared using the Wizard kit from Promega UK Ltd; Southampton SO 16 7NS, UK and sequenced by the Sanger-Coulson method (F Sanger, S Nicklen, A R Coulson; “DNA sequencing with chain terminating inhibitors”; Proceedings of the National Academy of Sciences of the USA 1977; 74: 5463-5467). V region sequences were compared with available sequences of human Ig genes using Ig blast (www.ncbi.nlm.nih.gov/igblast/igblast.cgi).
Immunoprecipitation Assay (IPA)
The cDNA encoding full length TSH receptor was placed downstream of the T7 promoter in pYES2 (Invitrogen) and used in an in vitro TnT system (Promega UK Ltd) to produce TSH receptor labelled with 35S-methionine as previously described (L Prentice, J Sanders, M Perez, R Kato, J Sawicka, Y Oda, D Jaskolski, J Furmaniak, B Rees Smith; “Thyrotropin (TSH) receptor autoantibodies do not appear to bind to the TSH receptor produced in an in vitro transcription/translation system”; Journal of Clinical Endocrinology and Metabolism 1997; 82: 1288-1292). Briefly 50 μL, 35S-labelled TSH receptor (25 000-30 000 cpm) diluted in HSB (150 mmol/L Tris-HCL pH 8. 3.200 mmol/L NaCl and 10 mg/mL bovine serum albumin containing 1% Tween 20) were added to duplicate 50 μL aliquots of diluted test sample and incubated for 2 hours at room temperature. Immune complexes were then precipitated by addition of protein A sepharose (Sigma-Aldrich) and counted in a scintillation counter.
TSH Receptor Preparations and Western Blotting
Full-length human TSH receptor was expressed in CHO-K1 cells, extracted with 1% Triton X-100 and purified by TSH receptor monoclonal antibody affinity chromatography as described previously (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; 10:1051-1059).
The purified CHO cell produced TSH receptor was run on 9% SDS-PAGE gels, blotted onto nitrocellulose and reacted with test antibodies as described previously (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: 1051-1059).
Epitope Analysis Using TSH Receptor Peptides
Twenty six peptides each 25aa long covering the whole of the extracellular domain of the human TSH receptor were kindly provided by Dr J Morris (J C Morris, E R Bergert, D J McCormick; “Structure-function studies of the human thyrotropin receptor. Inhibition of binding of labeled thyrotropin (TSH) by synthetic human TSH receptor peptides”; Journal of Biological Chemistry 1993; 268: 10900-10905). A human 21-OH peptide (C1, SSSRVPYKDRARLPL) which binds to an M21-OH5 MAb (S Chen, J Sawicka, L Prentice, J F Sanders, H Tanaka, V Petersen, C Betterle, M Volpato, S Roberts, M Powell, B Rees Smith, J Furmaniak; “Analysis of autoantibody epitopes on steroid 21-hydroxylase using a panel of monoclonal antibodies”; Journal of Clinical Endocrinology and Metabolism 1998; 83: 2977-2986) was used as a positive control and a human monoclonal antibody to GAD65 (N Hayakawa, L D K E Premawardhana, M Powell, M Masuda, C Arnold, J Sanders, M Evans, S Chen, J C Jaume, S Baekkeskov, B Rees Smith, J Furmaniak; “Isolation and characterization of human monoclonal autoantibodies to glutamic acid decarboxylase”; Autoimmunity 2002; 35: 343-355) was used as a negative control. The peptide ELISA was carried out as described previously (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: 1051-1059).
Interaction of Monoclonal TSHR Autoantibody Preparations with the TSH Receptor Coated onto Plastic Tubes or ELISA Plate Wells
(a) 125I-Labelled Autoantibody
Test samples including patient sera (100 μL) were incubated in TSH receptor coated tubes (RSR Ltd.) 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 labelled autoantibody preparation (30,000 cpm) and incubation at room temperature for 1 hour with shaking. The tubes were then washed twice with 1 mL of assay buffer, aspirated and counted in a gamma counter. Inhibition of 125I-labelled autoantibody binding was calculated using the formula as for inhibition of TSH binding (see above).
(b) Biotin Labelled Monoclonal Autoantibody and Biotin Labelled TSH
The procedure described previously (J Bolton, J Sanders, Y Oda, C Chapman, R Konno, J Furmaniak and B Rees Smith; “Measurement of thyroid-stimulating hormone receptor autoantibodies by ELISA”; Clinical Chemistry, 1999; 45: 2285-2287) was used. Briefly, test samples including patient sera (75 μL) were incubated in TSH receptor coated ELISA plate wells (RSR Ltd) for 2 hours with shaking (200 shakes per minute) on an ELISA plate shaker. Test samples were then removed and the wells washed once with assay buffer. Biotin-labelled monoclonal TSH receptor autoantibody (1 ng in 100 μL) or biotin labelled porcine TSH (RSR Ltd; 5 ng in 100 μL) were then added and incubation continued for 25 minutes at room temperature without shaking. The wells were washed once, 100 μL of streptavidin-peroxidase (RSR Ltd; 10 ng in 100 μL) added and incubation continued for 20 minutes at room temperature without shaking. The wells were then washed 3 times, the peroxidase substrate tetramethyl benzidine (RSR Ltd, 100 μL) added. After incubation for 30 minutes at room temperature without shaking 50 μL of 0.5M H2SO4 was added to stop the substrate reaction and the absorbance of each well read at 450 nm on an ELISA plate reader. Inhibition of biotinylated MAb or TSH binding was expressed as an index calculated as:—
Scatchard Analysis of Monoclonal Autoantibody Binding to TSH Receptor Coated Tubes
Unlabelled IgG or Fab in 50 μL of assay buffer and 50 μL of 125I-labelled hMAb IgG or Fab (30,000 cpm in assay buffer) were incubated for 2 hours at room temperature with gentle shaking, washed twice with 1 mL of assay buffer and counted in a gamma counter. The concentration of IgG or Fab bound vs bound/free was plotted (G Scatchard; “The attraction of proteins for small molecules and ions”; Annals of the New York Academy of Sciences 1949; 51: 660-672) to derive the association constants.
Binding of TSH Receptor to Tubes Coated with Monoclonal TSH Receptor Autoantibodies
Test samples including patient sera (100 μl) and detergent solubilised TSH receptor (20 μL) were incubated for 1 hour at room temperature. Duplicate 50 μL aliquots of the incubation mixture were then added to plastic tubes (Nunc Maxisorp) which had been coated with monoclonal TSH receptor autoantibody Fab (200 μL of 10 μg/mL overnight at 4° C. followed by washing and post coating). After incubation for 1 hour at room temperature with gentle shaking, the tubes were washed, 100 μL (40,000 cpm) of 125I-labelled TSH receptor C terminal monoclonal antibody 4E31 (J Sanders, Y Oda, 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: 3797-3802) added and incubation continued for a further 1 hour with gentle shaking. Then tubes were washed and counted for 125I.
Cloning and Expression of Recombinant hMAb TSHR1 Fab in E. coli
The hMAb TSHR1 heavy chain RT-PCR product (see Variable Region Gene Analysis section) was cut with XhoI and Spe1 restriction endonucleases and the hMAb TSHR1 light chain PCR product was cut with Sad and XbaI restriction endonucleases and both heavy and light chain cDNAs cloned into the Immunozap H/L vector (Stratagene Europe; Amsterdam, Netherlands) (I Matthew, G Sims, S Ledwidge, D Stott, D Beeson, N Willcox, A Vincent; “Antibodies to acetylcholine receptor in parous women with myasthenia: evidence for immunization by fetal antigen”; Laboratory Investigation 2002; S2: 1-11) under the control of the lacZ promoter. Plasmid DNA was prepared using the Qiagen midi plasmid purification kit (Qiagen Ltd; Crawley, RH10 9AX, UK) and the presence of hMAb TSHR1 heavy and light chain cDNAs confirmed by sequencing using the Sanger-Coulson method (F Sanger, S Nicklen, A R Coulsen; “DNA sequencing with chain terminating inhibitors”; Proceedings of the National Academy of Sciences of the USA 1977; 74: 5463-5467). Plasmid DNA was transformed into 2 different E. coli strains (a) XL1-Blue MRF′ (Stratagene) and (b) HB2151 (Amersham Biosciences) and grown overnight at 37° C. on LB ampicillin (Tryptone 10 g/L, Yeast Extract 5 g/L, NaCl 10 g/L, 100 μg/mL final concentration Ampicillin) agar plates (15 g/L agar). Precultures (one colony in 3 mL LB ampicillin+1% glucose) were grown overnight at 37° C. with shaking. Production of the recombinant Fab is inhibited in the presence of glucose. Precultures after overnight incubation were diluted 1/100 (0.5 mL in 50 mL LB ampicillin) and grown at 37° C. until the OD600 was between 0.4-0.6. These cultures were placed at 30° C. with shaking for 20 minutes. Thereafter isopropyl-β-D thiogalactoside (IPTG) was added to a final concentration of 1 mmol/L and cultures continued to be incubated overnight (16 hours) at 30° C. with shaking. The cultures were then centrifuged at 3000 rpm for 30 minutes at 4° C. and the culture supernatants and pellets recovered. The pellets, were resuspended in 1 mL of ice cold TES buffer (0.2 mol/L Tris-HCl pH 8.0, 0.5 mol/L EDTA and 0.5 mol/L sucrose) by vortexing. A further 1.5 mL of ice cold TES buffer diluted 5× in H2O was added, the mixture vortexed again and incubated on ice for 30 minutes then recentrifuged to give a second supernatant or periplasmic fraction (PF). The culture supernatant and PF were filtered through a 0.45 μm filter and dialysed overnight into 10 mmol/L Tris pH 7.5, 50 mmol/L NaCl. Culture supernatant or PF from untransformed XL1-Blue MRF′ and HB2151 cells and XL1-Blue MRF′ transformed with hMAb TSHR1 plasmid (XL1-Blue MRF′/hMAb TSHR1) and HB2151 transformed with hMAb TSHR1 plasmid (HB2151/hMAb TSHR1) grown with glucose without IPTG ie non-induced were also prepared. The culture supernatants and PF were assayed for (a) their ability to inhibit TSH binding to the TSHR, (b) their ability to stimulate cyclic AMP production in CHO cells expressing TSHR, and (c) total recombinant Fab concentration by radioimmunoassay. In this assay, calibrators and test materials including culture supernatants and PF (100 μL in duplicate) diluted in assay buffer (50 mmol/L NaCl, 10 mmol/L Tris-HCl pH 7.8, 0.1% Triton X-100) were incubated for 1 hour at room temperature in plastic tubes coated with Fab specific goat anti human IgG (from Sigma Aldrich, Poole, BH12 4QH, UK). The tubes were then washed with assay buffer (2×1 mL) and 100 μL of 125I-labelled hMAb TSHR1 Fab (30,000 cpm) added followed by incubation at room temperature. After 1 hour, the tubes were washed again (2×1 mL) and counted for 125I. The counts bound were plotted against concentration of Fab (hybridoma produced hMAb TSHR1 Fab) in the calibrators (5-500 ng/mL) and the concentration of recombinant Fab in the various test materials read off this calibration curve. The detection limit for this assay was 5-10 ng/mL of Fab.
Cloning and Expression of Recombinant 4B4 (a Human MAb to Glutamic Acid Decarboxylase or GAD) and Recombinant Hybrid Fabs (Mixed HC and LC of hMAb TSHR1 and 4B4)
Recombinant 4B4 Fab (4B4 is described in detail by N Hayakawa, LDKE Premawardhana, M Powell, M Masuda, C Arnold, J Sanders, M Evans, S Chen, J C Jaume, S Baekkeskov, B Rees Smith, J. Furmaniak. Isolation and characterization of human monoclonal antibodies to glutamic acid decarboxylase. Autoimmunity 2002 volume 35 pp 343-355) and recombinant hybrid Fabs were produced by cloning the respective HC and LC into Immunozap H/L vector and expressed in HB2151 cells as described for recombinant hMAb TSHR1 Fab. The culture supernatants and periplasmic fraction were assayed for their ability to (a) inhibit TSH binding to the TSHR (b) to stimulate cyclic AMP production in CHO cells expressing the TSHR and (c) for total recombinant Fab concentration as described above. In addition, GAD Ab activity was assessed as described below.
Measurement of Recombinant GAD Ab Fab Activity in Culture Supernatants and Periplasmic Fractions
An assay based on the ability of GAD Ab Fab preparation to inhibit the binding of 125I-labelled GAD (from RSR Ltd, Cardiff, CF23 SHE, UK) to the human monoclonal antibody to GAD (4B4) was used. In the assay, test samples diluted in GAD Ab assay buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl pH 8.0, 1% v/v Tween 20, 1 g/L bovine serum albumin and 0.5 g/L NaN3) were incubated (50 μL in duplicate with 125I-labelled GAD (30,000 cpm in 50 μL of GAD Ab assay buffer) for 1 hour at room temperature. Then, 50 μL of 4B4 IgG (0.1 μg/mL in GAD Ab assay buffer) was added and incubation continued for 24 hours at room temperature. Solid phase protein A (50 μL in GAD Ab assay buffer; from RSR Ltd) was then added to precipitate complexes of 4B4 IgG-125I-labelled GAD (protein A does not react with complexes of Fab and 125I-labelled GAD). After allowing the reaction with protein A to proceed for 1 hour at room temperature, the precipitates were pelleted by centrifugation (1500 g for 30 minutes at 4° C.), washed with 1 mL of GAD Ab assay buffer and counted for 125I. 125I-labelled GAD binding in the absence of 4B4 IgG was 4-5% of the total cpm added.
Production of Anti-Idiotypic Antibodies to hMAb TSHR1
6-8 week old BALB/c mice were immunised intraperitoneally with 50 μg hMAb TSHR1 Fab in complete Freunds adjuvant followed by a second injection with 50 μg hMAb TSHR1 Fab in incomplete Freunds adjuvant after 25 days and a further injection of 50 μg hMAb TSHR1 Fab 4 days before removal of the spleen. The spleen cells from antibody positive mice (see below) were fused with a mouse myeloma cell line and monoclonal antibodies isolated as above for TSHR MAbs. The levels of anti-idiotypic antibody in the mouse sera and cell culture wells were measured by inhibition of 125I-hMAb TSHR1 Fab binding to TSHR coated tubes. In particular, duplicate 60 μL aliquots of test sample (diluted in assay buffer: 50 mmol/L NaCl, 10 mmol/L Tris-HCl pH 7.8, 0.1% Triton X-100) were incubated with 60 μL of 125I-hMAb TSHR1 Fab (30000 cpm diluted in assay buffer) for 1 hour at room temperature. 100 μL of the mixture was transferred to duplicate TSHR coated tubes (RSR Ltd) with 20 μL start buffer (see above) and incubation continued for a further two hours at room temperature with shaking. The tubes were then washed with 2×1 mL of assay buffer and counted for 125I. The presence of anti-idiotypic antibodies reactive with hMAb TSHR1 was evident from the ability of test samples to inhibit the binding of labelled hMAb TSHRI Fab to the TSHR coated tubes.
Results
Lymphocytes (30×106) obtained from 20 mL of patient's blood were plated out at 1×106 per well on a 48 well plate with 200 μL of EBV supernatant on feeder layers of mouse macrophages. On day 11 post EBV infection the supernatants were monitored for inhibition of 125I-TSH binding. One well was found to be positive for inhibition of binding, the levels of inhibition increasing to greater than 90% inhibition by day 16 and stayed at that level until day 24, after which time they decreased. The cultures were expanded and fused with K6H6/B5 cells on day 21, 23, 26 and 27 post EBV infection; in total 7 fusion experiments were carried out. Each fusion was plated across 3×96 well plates (ie 21 plates in total) and one well, stably producing antibodies with 125I-TSH binding inhibiting activity was obtained. This was followed by 3 rounds of re-cloning to yield a single clone producing a human monoclonal antibody which inhibited labelled TSH binding to the TSH receptor. This human monoclonal TSH receptor autoantibody was designated hMAb TSHR1 and was of subclass IgG1 with a lambda light chain.
The ability of different concentrations of hMAb TSHR1 IgG and Fab to inhibit labelled TSH binding to the TSH receptor is shown in
125I-labelled hMAb TSHR1 IgG bound to TSH receptor coated tubes and Scatchard analysis indicated an association constant of 5×1010 molar−1. This binding was inhibited by sera from patients with Graves' disease who had TSH receptor autoantibodies (detectable by inhibition of labelled TSH binding) (Table 1), 125I-labelled hMAb TSHR1 Fab also bound to TSH receptor coated tubes (association constant by Scatchard analysis=4.5×1010 molar−1) and this binding was inhibited by TSH receptor autoantibody positive Graves' sera (Table 2). In addition, detergent solubilised preparations were able to bind to plastic tubes coated with hMAb TSHR1 and this binding could be inhibited by sera containing TSH receptor autoantibodies (Table 3).
As shown in Table 4 hMAb TSHRI-biotin bound to TSH receptor coated ELISA plates and the binding was inhibited by the international reference preparation NIBSC 90/672 and serum from patients with Graves' disease. Inhibition of binding was not observed by sera from healthy blood donors.
hMAb TSHR1 IgG did not react with full length TSH receptor preparations on Western blot analysis nor did it react well with 35S-labelled full length TSH receptor in the immunoprecipitation assay nor in the TSH receptor peptide ELISA. This lack of reactivity indicates that hMAb TSHR1 reacts with conformational rather than linear epitopes on the TSH receptor.
Sequence analysis of the genes coding for hMAb TSHR1 indicated that the heavy chain V region genes were of the VH5 family, the D gene of the D6-13 family and the J gene of the JH5 family and for the light chain the V-gene region is from the VL1-11 germline and the J-gene region is from the JL3b germline. The heavy chain nucleotide and amino acid sequences are shown in
Comparison of the activities of hMAb TSHR1 IgG preparations and the international standard for TSH receptor autoantibodies in terms of inhibition of labelled TSH binding are shown in Table 5. This enabled a specific activity of hMAb TSHR1 IgG to be estimated as 138 units of NIBSC 90/672 per mg of protein when the assays were carried out in serum and 163 units per mg when the assays were carried out in assay buffer (mean of the 2 values=150 units/mg). hMAb TSHR1 Fab preparations were 288 and 309 units per mg in serum and assay buffer respectively (mean of the 2 values=300 units/mg). Table 6 shows a similar analysis of the lymphocyte donor serum and the donor serum IgG. As can be seen the donor serum contains a mean of 0.38 units/mL of NIBSC 90/672 (0.36 and 0.4 in serum and assay buffer respectively) and the donor serum IgG has a mean specific activity of 0.059 units per mg of protein. These results are summarised in Table 7 and comparison with the specific activity of hMAb TSHR1 IgG (150 units/mg) indicates that the monoclonal autoantibody IgG is 2500 times more active than the lymphocyte donor serum IgG in terms of inhibition of TSH binding.
Initial assessment of the activities of the various IgG and serum preparations in terms of stimulation of cyclic AMP in CHO cells transfected with the TSH receptor are also shown in Table 7. The stimulation of cyclic AMP assay is characterized by considerable within assay and between assay variability. This relates to several factors including variation in the number and quality of cells initially seeded into the 96 well plates and variation in growth rate of the seeded cells over the subsequent 48 hours. Consequently the assays of hMAb TSHR1 IgG and Fab, lymphocyte donor serum and serum IgG and NIBSC 90/672 were carried out repeatedly and the results are summarized in Table 8. The specific activity of the hMAb TSHR1 IgG was 318 units per mg in the stimulation assay compared with 0.1 units per mg for the lymphocyte donor serum IgG ie the monoclonal autoantibody IgG was about 3000 times as active as the donor serum IgG in terms of stimulation of cyclic AMP production. This value is in reasonable agreement with the value of 2500 times observed for inhibition of TSH binding measurements (see above and Tables 5 and 6). Table 9 shows a further analysis of the TSH receptor stimulating effects of hMAb TSHR1 IgG and Fab and the lymphocyte donor serum IgG.
The effects of porcine TSH and hMAb TSHR1 IgG on stimulation of cyclic AMP production in CHO cells expressing the TSH receptor were additive as can be seen in the results shown in Table 10.
Typical results observed in the stimulation of cyclic AMP assay with the reference preparation NIBSC 90/672 are shown in Table 11.
Tables 12 and 13 show the effects of the various E. coli culture supernatants in terms of inhibition of labelled TSH binding and stimulation of cyclic AMP production respectively. Transformed (with hMAb THSR1 plasmid) and IPTG induced cultures of both strains of E. coli produced sufficient amounts of recombinant hMAb TSHR Fab to act as potent inhibitors of TSH binding (Table 12) and powerful stimulators of cyclic AMP production (Table 13). Control culture supernatants (from untransformed cells and transformed but non-induced cells) did not produce detectable levels of binding inhibition (Table 12) or stimulating (Table 13) activities.
In further control experiments, a recombinant human antibody Fab produced by cloning and expression of the HC and LC of a human monoclonal antibody to GAD (4B4) were analysed. Assays of culture supernatants and periplasmic fractions indicated that recombinant 4B4 Fab did not have detectable inhibition of TSH binding activity (Tables 14 and 15) or TSHR stimulating activity (Tables 16 and 17). Furthermore, hybrid Fabs consisting of (a) hMAb TSHR1 HC and 4B4 LC (b) hMAb TSHR1 LC and 4B4 HC did not show interaction with the TSHR in either assay (Tables 14-17). Assays for GAD Ab activity in these various recombinant Fab preparations were only able to detect GAD Ab expression in cells transformed with 4B4 HC and 4B4 LC (Tables 18 and 19). Recombinant hMAb TSHR1 Fab did not show detectable GAD Ab activity and neither did recombinant Fab hybrids consisting of mixtures of 4B4 and hMAb TSHR1 HC and LC (Tables 18 and 19).
The ability of hMAb TSHR1 to stimulate cyclic AMP production in CHO cells transfected with the TSHR was inhibited by patient sera containing TSHR autoantibodies which acted as TSH antagonists (
Immunisation of mice with hMAb TSHR1 Fab resulted in production of antibodies in the mice sera (polyclonal antibodies) which were capable of binding to hMAb TSHRI Fab in such a way as to inhibit the ability of the Fab to bind to the TSHR (Table 26). Furthermore, a monoclonal antibody produced from the spleen cells of a mouse immunised with hMAb TSHR1 Fab was also able to inhibit the Fab binding to the TSHR (Table 27).
Overall our analysis indicates that the human monoclonal autoantibody hMAb TSHR I has the TSH receptor binding and thyroid stimulating characteristics of the TSH receptor autoantibodies in the serum of the lymphocyte donor. As detailed above, the monoclonal antibody was also produced as a recombinant Fab preparation.
125I-labelled TSH binding to the TSH receptor
125I-labelled TSH binding to the TSH receptor
1TSHR binding was detected using a 125I-labelled mouse monoclonal antibody to the TSHR C terminus; total cpm = 39,000 per tube.
1Pool of healthy blood donor serum, 14.9% of total cpm bound to the TSHR in the presence of this serum pool only. 14.7% of total cpm bound to the TSHR in the presence of buffer only.
22G4 is a human monoclonal autoantibody to thyroid peroxidase.
1Pool of healthy blood donor serum, 14.7% of total cpm bound to the TSHR in the presence of this serum pool only. 16.3% of total cpm bound to the TSHR in the presence of buffer only.
2Units shown are NIBSC 90/672 international TSHR autoantibody reference preparation.
1Units shown are NIBSC 90/672.
2Values are a mean of results obtained in serum and in assay buffer (see Tables 5 and 6)
12G4 is a human monoclonal autoantibody to thyroid peroxidase
1Values shown are means of closely agreeing duplicate determinations
1Assay buffer = 50 mmol/L NaCl, 10 mmol/L Tris-HCl pH 7.8
2All dilutions were in assay buffer
3Inhibition of binding was calculated using the formula:
where A = binding in the presence of test sample
E coli strains (XL1-Blue MRF′ and HB2151 cells)
1Assay buffer = Hanks' buffered salt solution (NaCl free) containing 1 g/L glucose, 20 mmol/L HEPES, 222 mmol/L sucrose, 15 g/L bovine serum albumin (BSA) and 0.5 mmol/L 2 isobutyl-1-methyl xanthine pH 7.4
2Dilutions in assay buffer
3Basal = cAMP produced in the presence of assay buffer only
125I-TSH
1Inhibition of binding was calculated using the formula:
where A = % 125I-TSH binding in the presence of test sample
aDetection of TSHR Ab activity in the non-induced cells was due to constitutive activity of the promoter giving low level expression of Fab in the absence of IPTG.
1see footnote to Table 14
1Assay buffer: Hanks' buffered salt solution (NaCl free) containing 1 g/L glucose, 20 mmol/L HEPES, 222 mmol/L sucrose, 15 g/L bovine serum albumin (BSA) and 0.5 mmol/L 2 isobutyl-1-methyl xanthine pH 7.4
2Dilutions in assay buffer
3Basal = cyclic AMP production in the presence of assay buffer only
aDetection of cyclic AMP stimulation activity in the non-induced cells was due to constitutive activity of the promoter giving low level expression of Fab in the absence of IPTG. Total recombinant Fab levels were undetectable (detection limit = 5-10 ng/mL) whereas the stimulation of cyclic AMP assay can detect as little as 0.3 ng/mL of hMAb TSHR1 Fab.
1,2,3see footnote to Table 16
aDetection of TSHR Ab activity in the non-induced cells was due to constitutive activity of the promoter giving low level expression in the absence of IPTG.
125I-GAD
1Inhibition of binding was calculated using the formula:
where A = 125I-GAD binding in the presence of test sample
1see footnote to Table 18
1All dilutions were made in assay buffer (Hanks' buffered salt solution (NaCl free) containing 1 g/L glucose, 20 mmol/L HEPES, 222 mmol/L sucrose, 15 g/L bovine serum albumin (BSA) and 0.5 mmol/L 2 isobutyl-1-methyl xanthine pH 7.4)
2Basal = cyclic AMP production in the presence of assay buffer only
32G2 is a mouse monoclonal antibody to thyroglobulin (100 μg/mL of IgG preparation)
42B4 and 9D33 are mouse monoclonal antibodies to the TSHR (100 μg/mL of IgG preparations)
1,2,4See footnotes for Table 20. In a separate experiment a control mouse MAb to thyroglobulin (2G2 IgG 100 μg/mL) was shown to have no effect on pTSH (0.5 ng/mL) stimulation of cyclic AMP production (pTSH plus assay buffer = 12.7 × basal; pTSH plus 2G2 = 11.7 × basal)
125I-9D33 binding to TSHR coated tubes
125I-hMAb
1251-9D33
1All dilutions were in a pool of healthy blood donor sera (HBD pool)
2Inhibition of binding was calculated using the formula:
where A = % binding in the presence of test sample
125I-9D33 bound
125I-9D33
125I-TSH
1HBD pool = pool of healthy blood donor sera
2Inhibition of binding was calculated using theformula:
where A = % binding in the presence of test serum
125I-9D33 bound
125I-9D33 binding (%)2
1HBD pool = pool of healthy blood donor sera, test sera were diluted in this pool sera A and B have TSH antagonist activity sera C and D have TSH agonist activity
2Inhibition of binding was calculated with the formula used in Table 23
125I-9D33 bound
125I-9D33
125I-TSH
190/672 diluted in a pool of healthy blood donor sera
2Inhibition of binding was calculated with the formula used in Table 23
1Test samples diluted in assay buffer. Binding in the presence of assay buffer was 43%
2lnhibition of binding was calculated using the formula:
where A = % 125I-hMAb TSHR1 Fab binding in the presence of test sample
125I-hMAb TSHR1
1see footnote to Table 26
| Number | Date | Country | Kind |
|---|---|---|---|
| 0227964.4 | Nov 2002 | GB | national |
| 0302140.9 | Jan 2003 | GB | national |
| 0315147.9 | Jun 2003 | GB | national |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5565332 | Hoogenboom et al. | Oct 1996 | A |
| 5840853 | Segre et al. | Nov 1998 | A |
| 6537760 | Bergmann et al. | Mar 2003 | B1 |
| Number | Date | Country |
|---|---|---|
| 0719858 | Jul 1996 | EP |
| 1078986 | Feb 2001 | EP |
| 9109137 | Jun 1991 | WO |
| WO 9109137 | Jun 1991 | WO |
| WO 9826294 | Jun 1998 | WO |
| 9964865 | Dec 1999 | WO |
| 03018632 | Mar 2003 | WO |
| Entry |
|---|
| Oda et al., (J Mol Endocrinology. Apr. 1998; 20(2):233-44) (cited on Applicant's IDS filed Feb. 15, 2012). |
| Akamizu et al., (Endocrinology. Apr. 1999;140(4):1594-1601) (cited on Applicant's IDS filed Feb. 15, 2012). |
| Kohn et al., (J Clin Endo and Metab. 1997;82(12):3998-4009) (cited on Applicant's IDS filed Feb. 15, 2012). |
| UniProt, Accession No. P16473 (sequence version 1), Aug. 1, 1990. |
| Akamizu, T. et al., Characterization of Recombinant Monoclonal Antithyrotropin Receptor Antibodies (TSHRAbs) Derived from Lymphocytes of Patients with Graves' Disease: Epitope and Binding Study of Two Stimulatory TSHRAbs*, Endocrinology, 1999, pp. 1594-1601, vol. 140, No. 4. |
| Akamizu, T. et al., Molecular Analysis of Stimulatory Anti-Thyrotropin Receptor Antibodies (TSAbs) Involved in Graves' Disease, Journal of Immunology, 1996, pp. 3148-3152, vol. 157. |
| Chazenbalk, G. D. et al, Thyroid-stimulating autoantibodies in Graves disease preferentially recognize the free A subunit, not the thyrotropin holoreceptor, Journal of Clinical Investigation, 2002, pp. 209-217, vol. 110, No. 2. |
| Cundiff, J. G. et al., Studies Using Recombinant Fragments of Human TSH Receptor Reveal Apparent Diversity in the Binding Specificities of Antibodies That Block TSH Binding to Its Receptor or Stimulate Thyroid Hormone Production, the Journal of Clinical Endocrinology & Metabolism, 2001, pp. 4254-4260. |
| Kohn, L. D. et al., Characterization of Monoclonal Thyroid-Stimulating and Thyrotropin Binding-Inhibiting Autoantibodies from a Hashimotos Patient Whose Children Had Intrauterine and Neonatal Thyroid Disease, Journal of Clinical Endocrinology and Metabolism, 1997, pp. 3998-4009, vol. 82, No. 12. |
| Bulow Pedersen, I. et al., TSH-receptor antibody measurement for differentiation of hyperthyroidism into Graves' disease and multinodular toxic goitre: a comparison of two competitive binding assays, Clinical Endocrinology, 2001, pp. 381-390, vol. 55. |
| Sanders, J. et al., The Interaction of TSH Receptor Autoantibodies with 125 I-Labelled TSH Receptor, The Journal of Clinical Endocrinology & Metabolism, 1999, pp. 3797-3802, vol. 84, No. 10. |
| Shepherd, P.S. et al., Identification of an important thyrotrophin binding site on the human thyrotrophin receptor using monoclonal antibodies, Molecular and Cellular Endocrinology, 1999, pp. 197-206, vol. 149. |
| Van Der Heijden, J. H. W. et al., Limitations of the semisynthetic library approach for obtaining human monoclonal autoantibodies to the thyrotropin receptor of Graves' disease, Clin Exp Immunol, 1999, pp. 205-212, vol. 118. |
| Valente, L. A. et al., Monoclonal antibodies to the thyrotropin receptor: Stimulating and blocking antibodies derived from the lymphocytes of patients with Graves disease, Proc Natl Acad. Sci USA, 1982, pp. 6680-6684, vol. 79. |
| Yoshida, T. et al, Monoclonal Antibodies to the Thyrotropin Receptor Bind to a 56-kDa Subunit of the Thyrotropin Receptor and Show Heterogeneous Bioactivities*, The Journal of Biological Chemistry, 1988, pp. 16341-16347, vol. 263, No. 31. |
| Zhong, X. et al., Cloning and sequence analysis of light variable region gene of anti-human retinoblastoma monoclonal antibody, Yan Ke Xue Bao, 2002, pp. 185-189, vol. 18, No. 3 (Abstract). |
| Saper et al., Magic Peptides, Magic Antibodies: Guidelines for Appropriate Controls for Immunohistochemistry, The Journal of Comparative Neurology, 2003, pp. 161-163, vol. 465. |
| NCBI Accession No. M32215, Misrahi, M. et al., Cloning, sequencing and expression of human TSH receptor, 1995. |
| Costagliola, et al., Monoclonal Antibodies with Thyroid Stimulating Activity, at Last, Thyroid, 2002, pp. 1039-1041, vol. 12, No. 12. |
| Ando, T. et al., Monoclonal antibodies to the thyrotropin receptor, Clin Devel Immunol, 2005, pp. 137-143, vol. 12, No. 2. |
| Ando, T. et al., A monoclonal thyroid-stimulating antibody, the Journal of Clinical Investigation, 2002, pp. 1667-1674, vol. 110, No. 11. |
| Sanders, J. et al., Thyroid-Stimulating Monoclonal Antibodies, Thyroid, 2002, pp. 1043-1050, vol. 12, No. 12. |
| Rapoport, B. et al., The Thyrotropin (TSH) Receptor: Interaction with TSH and Autoantibodies, Endocrine Reviews, 1998, (downloaded from edrv.endojournals.org by on Apr. 18, 2008), pp. 673 and 698, vol. 19, No. 8. |
| Chen, S. et al., Analysis of Autoantibody Epitopes on Steroid 21-Hydroxylase Using a Panel of Monoclonal Antibodies, Journal of Clinical Endocrinology and Metabolism, 1998, pp. 2977-2986, vol. 83, No. 8. |
| Collet, T.A. et al., A binary plasmid system for shuffling combinatorial antibody libraries, Proc. Natl. Acad. Sci. USA, 1992, pp. 10026-10030, vol. 89. |
| Latif, R. et al., Oligomerization of the Human Thyrotropin Receptor, The Journal of Biological Chemistry, 2001, pp. 45217-45224, vol. 276, No. 48. |
| Myers, M. A. et al., Conformational Epitopes on the Diabetes Autoantigen GAD65 Identified by Peptide Phage Display and Molecular Modeling, The Journal of Immunology, 2000, pp. 3830-3838, vol. 165. |
| Morris, J. C. et al., Structure-Function Studies of the Human Thyrotropin Receptor, the Journal of Biological Chemistry, 1993, pp. 10900-10905, vol. 268, No. 15. |
| Bolton, J. et al., Measurement of Thyroid-stimlating Hormone Receptor Autoantibodies by ELISA, Clinical Chemistry, 1999, pp. 2285-2287, vol. 45, No. 12. |
| Oda, Y. et al., Binding characteristics of antibodies to the TSH receptor, Journal of Molecular Endocrinology, 1998, pp. 233-244, vol. 20. |
| Prentice, L. et al., Thyrotropin (ISH) Receptor Autoantibodies Do Not Appear to Bind to the TSH Receptor Produced in an in Vitro Transcription/Translation System, Journal of Clinical Endocrinology and Metabolism, 1997, pp. 1288-1292, vol. 82, No. 4. |
| Sanger, F. et al., DNA sequencing with chain-terminating inhibitors, Proceedings of the National Academy of Sciences of the USA, 1977, pp. 5463-5467, vol. 74, No. 12. |
| Number | Date | Country | |
|---|---|---|---|
| 20120276117 A1 | Nov 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10537280 | US | |
| Child | 13339111 | US |
