No abstract

The properties of a human monoclonal antibody to the thyrotropin receptor (TSHR) (M22) with the characteristics of patient sera thyroid stimulating autoantibodies is described. Similar concentrations (pmol/L) of M22 Fab and porcine TSH had similar stimulating effects on cyclic adenosine monophosphate (cAMP) production in TSHR-transfected Chinese hamster ovary cells whereas higher doses of intact M22 immunoglobulin G (IgG) were required to cause the same level of stimulation. Patient sera containing TSHR autoantibodies with TSH antagonist (blocking) activity inhibited M22 Fab and IgG stimulation in a similar way to their ability to block TSH stimulation. Thyroid-stimulating monoclonal antibodies (TSmAbs) produced in mice inhibited 125I-TSH binding and 125I-M22 Fab binding to the TSHR but the mouse TSmAbs were less effective inhibitors than M22. These competition studies emphasized the close relationship between the binding sites on the TSHR for TSH, TSHR autoantibodies with TSH agonist activity, and TSHR autoantibodies with TSH antagonist activity. Recombinant M22 Fab could be produced in Escherichia coli and the recombinant and hybridoma produced Fabs were similarly active in terms of inhibition of TSH binding and cAMP stimulation. The crystal structure of M22 Fab was determined to 1.65 A resolution and is that of a standard Fab although the hypervariable region of the heavy chain protrudes further from the framework than the hypervariable region of the light chain. The M22 antigen binding site is rich in aromatic residues and its surface is dominated by acidic patches on one side and basic patches on the other in agreement with an important role for charge-charge interactions in the TSHR-autoantibody interaction.

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.

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.

SummaryConsiderable evidence has associated the expression of matrix metalloproteinases (MMPs) with the degradation of cartilage and bone in chronic conditions such as arthritis. Direct evaluation of MMPs' role in vivo has awaited the development of MMP inhibitors with appropriate pharmacological properties. We have identified butanediamide, N4-hydroxy-2-(2-methylpropyl)-as a potent MMP inhibitor with sufficient solubility and stability to permit evaluation in an experimental model of chronic destructive arthritis (adjuvant-induced arthritis) in rats. In this model, pronounced acute and chronic synovial inflammation, distal tibia and metatarsal marrow hyperplasia associated with osteoclasia, severe bone and cartilage destruction, and ectopic new bone growth are well developed by 3 wk after adjuvant injection. Rats were injected with Freund's adjuvant on day 0. GI168 was was administered systemically from days 8 to 21 by osmotic minipumps implanted subcutaneously. GI168 at 6, 12, and 25 mg/kg per d reduced ankle swelling in a dose-related fashion. Radiologicai and histological ankle joint evaluation on day 22 revealed a profound dose related inhibition of bone and cartilage destruction in treated rats relative to rats receiving vehicle alone. A significant reduction in edema, pannus formation, periosteal new bone growth and the numbers of adherent marrow osteoclasts was also noted. However, no significant decrease in polymorphonuclear and mononuclear leukocyte infiltration of synovium and marrow hematopoietic cellularity was seen. This unique profile of antiarthritic activity indicates that GI168 is osteo-and chondro-protective, and it supports a direct role for MMP in cartilage and bone damage and pannus formation in adjuvant-induced arthritis.