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1.
Thyroid ; 18(7): 735-46, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18631002

ABSTRACT

BACKGROUND: Human monoclonal autoantibodies (MAbs) are valuable tools to study autoimmune responses. To date only one human MAb to the thyrotropin (TSH) receptor (TSHR) with stimulating activity has been available. We now describe the detailed characterization of a blocking type human MAb to the TSHR. METHODS: A single heterohybridoma cell line was isolated from the peripheral blood lymphocytes of a patient with severe hypothyroidism (TSH 278 mU/L) using standard techniques. The line stably expresses a TSHR autoantibody (5C9; IgG1/kappa). Ability of 5C9 to bind and compete with 125I-TSH or TSHR antibodies binding to the TSHR was tested using tubes coated with solubilized TSHR. Furthermore, the blocking effects of 5C9 on stimulation of cyclic AMP production was assessed using Chinese hamster ovary (CHO) cells expressing the wild-type human TSHR or TSHRs with amino acid mutations. MAIN OUTCOME: 5C9 IgG bound to the TSHR with high affinity (4 x 10(10) L/mol) and inhibited binding of TSH and a thyroid-stimulating human monoclonal autoantibody (M22) to the receptor. 5C9 IgG preparations inhibited the cyclic AMP-stimulating activities of TSH, M22, serum TSHR autoantibodies and thyroid-stimulating mouse monoclonal antibodies. Furthermore 5C9 reduced the constitutive activity of wild-type TSHR and TSHR with some activating mutations. The effect of different amino acid mutations in the TSHR on 5C9 biological activity was studied and TSHR Lys129Ala or Asp203Ala completely abolished the ability of 5C9 to block TSH-mediated stimulation of cyclic AMP production. CONCLUSIONS: The availability of 5C9 provides new opportunities to investigate the binding and biological activity of TSHR blocking type autoantibodies including studies at the molecular level. Furthermore, monoclonal antibodies such as 5C9 may well provide the basis of new drugs to control TSHR activity including applications in thyroid cancer and Graves' ophthalmopathy.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Receptors, Thyrotropin/immunology , Thyroid Gland/drug effects , Adult , Animals , Antibodies, Monoclonal/therapeutic use , Autoantibodies/blood , CHO Cells , Cell Line , Cricetinae , Cricetulus , Cyclic AMP/metabolism , Female , Graves Ophthalmopathy/drug therapy , Humans , Hypothyroidism/metabolism , Mutation/genetics , Ovary/cytology , Ovary/drug effects , Ovary/metabolism , Receptors, Thyrotropin/genetics , Receptors, Thyrotropin/metabolism , Thyroid Gland/metabolism , Thyroid Neoplasms/drug therapy
2.
Thyroid ; 16(12): 1195-206, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17199429

ABSTRACT

The effects of an extensive series of mutations in the TSH receptor (TSHR) leucine-rich domain (LRD) on the ability of thyroid-stimulating monoclonal antibodies (TSMAbs) and TSH to bind to the receptor and stimulate cyclic AMP production in TSHR-transfected CHO cells has been investigated. In addition, the ability of a mouse monoclonal antibody with blocking (i.e., antagonist) activity (RSR-B2) to interact with mutated receptors has been studied. Several amino acids distributed along an extensive part of the concave surface of the LRD were found to be important for binding and stimulation by the thyroid-stimulating human MAb M22 but did not appear to be important for TSH binding and stimulation. Most of these amino acids important for M22 interactions were also found to be important for the stimulating activity of six different mouse TSMAbs and a hamster TSMAb. Furthermore, most of these same amino acids were important for stimulation by TSHR autoantibodies in a panel of sera from patients with Graves' disease. Amino acid R255 was the only residue found to be unimportant for TSH stimulation but critical for stimulation by all thyroid-stimulating antibodies tested (23 patient serum TSHR autoantibodies, M22, and all seven animal TSMAbs). About half the amino acids (all located in the N-terminal part of the LRD) found to be important for M22 activity were also important for the blocking activity of RSR-B2 and although the epitopes for the two MAbs overlap they are different. As the two MAbs have similar affinities, their epitope differences are probably responsible for their different activities. Overall our results indicate that different TSMAbs and different patient sera thyroid-stimulating autoantibodies interact with the same region of the TSHR, but there are subtle differences in the actual amino acids that make contact with the different stimulators.


Subject(s)
Antibodies, Monoclonal/metabolism , Receptors, Thyrotropin/genetics , Thyrotropin/metabolism , Animals , Antigen-Antibody Reactions , CHO Cells , Cricetinae , Cricetulus , Cyclic AMP/biosynthesis , Graves Disease/immunology , Humans , Mice , Models, Molecular , Point Mutation , Protein Structure, Tertiary , Receptors, Thyrotropin/metabolism , Thyrotropin/antagonists & inhibitors , Thyrotropin/physiology , Transfection
3.
Thyroid ; 12(12): 1043-50, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12593717

ABSTRACT

Thyrotropin (TSH) receptor monoclonal antibodies (TSHR mAbs) were obtained from cDNA-immunized NMRI mice. Three mAb immunoglobulin Gs (IgGs) (TSmAbs 1-3) that had distinct V(H )and V(L) region sequences stimulated cyclic adenosine monophosphate (cAMP) production in isolated porcine thyroid cells greater than 10x basal and as little as 20 ng/mL (0.13 nmol/L) of TSmAb 1 IgG caused a 2x basal stimulation. TSmAb 1 and 2 Fab fragments were also effective stimulators and thyroid-stimulating activities of the IgGs and Fabs were confirmed using TSHR transfected Chinese hamster ovary (CHO) cells. The TSmAbs also inhibited (125)I-labeled TSH binding to TSHR-coated tubes by 50% or more at concentrations of 1 microg/mL or less and gave 15%-20% inhibition at 20-50 ng/mL. (125)I-labeled TSmAbs bound to TSHR-coated tubes with high affinity (approximately 10(10) L/mol) and this binding was inhibited by TSHR autoantibodies with both TSH agonist and antagonist activities. Inhibition of labeled TSmAb binding by Graves' sera correlated well with inhibition of TSH binding (r = 0.96; n = 18; p < 0.001 for TSmAb 2). The TSmAbs have considerable potential as (1) new probes for TSHR structure-function studies, (2) reagents for new assays for TSHR autoantibodies, and (3) alternatives to recombinant TSH in various in vivo applications.


Subject(s)
Antibodies, Monoclonal/immunology , Graves Disease/immunology , Thyroid Gland/immunology , Animals , Animals, Outbred Strains , Antibodies, Monoclonal/pharmacology , Autoantibodies/immunology , Autoantibodies/pharmacology , Binding, Competitive/immunology , CHO Cells , Cricetinae , Humans , Immunization , Iodine Radioisotopes , Mice , Receptors, Thyrotropin/immunology , Receptors, Thyrotropin/metabolism , Thyrotropin/metabolism , Thyrotropin/pharmacology
4.
Thyroid ; 12(12): 1051-61, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12593718

ABSTRACT

A panel of monoclonal antibodies (mAbs) to the thyrotropin receptor (TSHR) was prepared using three different immunization strategies. The mAbs obtained (n = 138) reacted with linear epitopes covering most of the TSHR extracellular domain and with conformational epitopes. mAbs that bound to five different regions of the TSHR (amino acids [aa] 32-41, aa 36-42, aa 246-260, aa 277-296, and aa 381-385) were able to inhibit (125)I-labeled thyrotropin (TSH) binding to solubilized TSHR preparations. Fab and immunoglobulin G (IgG) preparations were similarly effective inhibitors for mAbs reactive with aa 246-260, aa 277-291 and aa 381-385 suggesting that these three regions of the TSHR are involved in TSH binding. In contrast mAbs reactive with aa 32-41 and aa 36-42 were not effective at inhibiting TSH binding when Fab preparations were used, suggesting that these N terminal regions of the TSHR were less critical for TSH binding. Our studies suggest that three distinct and discontinuous regions of the TSHR (aa 246-260 and 277-296 on the TSHR A subunit) and aa 381-385 (on the TSHR B subunit) fold together to form a complex TSH binding pocket. Alignment of the aa sequences of these three regions in TSHRs from different species indicates that they are highly conserved.


Subject(s)
Receptors, Thyrotropin/chemistry , Receptors, Thyrotropin/metabolism , Thyrotropin/metabolism , Animals , Animals, Outbred Strains , Antibodies, Monoclonal/pharmacology , Binding Sites/immunology , Binding, Competitive/immunology , Blotting, Western , CHO Cells , Cricetinae , Epitopes , Humans , Immunization , Iodine Radioisotopes , Mice , Mice, Inbred BALB C , Protein Structure, Tertiary , Receptors, Thyrotropin/immunology , Sulfur Radioisotopes
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