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 interaction between collagen, von Willebrand factor (VWF), and glycoprotein Ib is the first step in hemostasis and thrombosis especially under high shear conditions. We studied the inhibition of the VWF-collagen interaction by using an antihuman VWF monoclonal antibody 82D6A3 to prevent arterial thrombosis in baboons to develop a new kind of antithrombotic strategy and determine for the first time experimental in vivo data concerning the importance of the collagen-VWF interaction. We used a modified Folts model to study the antithrombotic efficacy of 82D6A3, where cyclic flow reductions (CFRs) were measured in the femoral artery. Administering a dose of 100, 300, and 600 g/kg resulted in a 58.3%, 100%, and 100% reduction in the CFRs, respectively. When 100 g/kg 82D6A3 was infused into the baboons, 80% of VWF-A3 domain was occupied, corresponding to 30% to 36% ex vivo inhibition of VWF binding to collagen, with no prolongation of the bleeding time. The bleeding time was also not significantly prolonged when the CFRs were abolished at doses of 300 g/kg and 600 g/kg. At these doses 100% of VWF was occupied by the antibody and 100% ex vivo inhibition of the VWF-collagen binding was observed. 82D6A3 has a high affinity for VWF; after 48 hours still 68% VWF (300g/kg) was occupied with a pharmacologic effect up to 5 hours after administration (80%-100% occupancy). In conclusion, these results clearly indicate that the VWFcollagen interaction is important in vivo in thrombosis under high shear conditions and thus might be a new target for preventing arterial thrombosis. (Blood. 2002;99:3623-3628)

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.