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.
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.
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)
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.
In this study we describe the first monoclonal antibody, integrin activated conformation-1 (IAC-1), to recognize the active form of the platelet-collagen receptor, the integrin α2β1. IAC-1 has the following properties: (1) IAC-1 fails to bind to resting platelets but readily interacts with platelets stimulated by the glycoprotein VI-specific agonist, convulxin, and by other agonists; (2) similar concentration response relationships for binding of IAC-1 and soluble collagen were observed in convulxin-stimulated platelets; (3) the epitope for IAC-1 is T199Y200K201, which is located at the opposite site of the metal ion-dependent adhesion site in a region not involved in the I-domain “shifts” that occur upon ligand binding; (4) IAC-1 strongly binds to recombinant α2 I-domain, therefore suggesting that the neo-epitope appears to be exposed by an “unmasking” of I-domain-covering regions upon activation; (5) IAC-1 binds to platelets during adhesion to collagen under shear conditions, demonstrating activation of α2β1; (6) as IAC-1 does not interfere with platelet-collagen binding, it defines a new class of antibodies that is distinct from those belonging to the “cation- and ligand-induced binding sites” (CLIBSs) and the “ligand mimetic” group. These characteristics make IAC-1 a very powerful tool to study α2β1 activation under dynamic and physiologically relevant conditions. (Blood. 2004;104:390-396)
Analysis of nine mouse monoclonal antibodies (mAbs) to the thyrotropin receptor (TSHR) with TSH antagonist activity showed that only one of the mAbs (RSR B2) was an effective antagonist of the human thyroid stimulating autoantibody M22. Crystals of B2 Fab were analyzed by x-ray diffraction and a crystal structure at 3.3 A resolution was obtained. The surface charge and topography of the B2 antigen binding site were markedly different from those of the thyroid-stimulating mAb M22 and these differences might contribute to the different properties of the two mAbs. B2 (but not other mouse TSHR-specific mAbs) was also an effective antagonist of thyroid stimulating autoantibody activity in 14 of 14 different sera from patients with Graves' disease. 125I-labeled B2 bound to the TSHR with high affinity (2 x 10(10) L/mol) and patient serum TSHR autoantibodies inhibited labeled B2 binding to the receptor in a similar way to inhibition of labeled TSH binding (r = 0.75; n = 20). Furthermore, labeled B2 binding was inhibited by patient serum TSHR autoantibodies with TSH antagonist activity and also by mouse and human thyroid stimulating mAbs. Overall, mAb B2 is a powerful antagonist of thyroid stimulating autoantibodies (and TSH) thus resembling closely patient serum TSH antagonist TSHR autoantibodies. Furthermore, B2 might have potentially important in vivo applications when tissues containing the TSHR (including those in the orbit) need to be made unresponsive to stimulating autoantibodies.
Monoclonal antibody (mAb) 82D6A3 is an anti-vonIn conclusion, to our knowledge, this is the first report where a modeled peptide containing a consensus sequence could be fitted onto the three-dimensional structure of the antigen, indicating that it might adopt the conformation of the discontinuous epitope.Platelet adhesion to subendothelial structures, more specifically to the thrombogenic compound collagen, is one of the first steps in a sequence of reactions that can lead to arterial thrombosis. Platelets interact with collagen both in a direct manner via their collagen receptors (e.g. ␣ 2  1 (1, 2) and glycoproteins IV (3) and VI (4, 5)) and indirectly with VWF, 1 forming the bridge between collagen and its platelet receptor glycoprotein Ib/IX/V (6). Binding via both ␣ 2  1 and VWF is necessary to sustain platelet adhesion under high shear forces (7-9); VWF-mediated interaction results in rolling of the platelets over the collagen surface (10), upon which the collagen receptors can interact with the damaged vessel wall, leading to firm adhesion. This is the result of platelet activation by the signal-transducing glycoprotein VI (11), leading to a gain-in-affinity of ␣ 2  1 (12) and activation of ␣ IIb  3 with platelet aggregation as a consequence.Both ␣ 2  1 and VWF bind to collagen through their I-domains, in VWF known as A-domains (13-19). A-domains form independent globular modules of some 200 amino acid residues. In VWF three such domains have been identified. The A1-domain contains the binding site for glycoprotein Ib (20,21), sulfatides (22), heparin (23), and collagen VI (24, 25), which constitutes the main reactive collagen in the extracellular matrix of endothelial cells. The A2-domain has no clear binding function but is sensitive to protease ADAMTS13-mediated enzymatic degradation (26,27), whereas the A3-domain (residues 920 -1111) contains the main binding site for fibrillar collagens such as type I and III (19,28). Recombinant A3-domain also binds to collagen (28), whereas deletion of A3 results in a VWF that binds 40 times less to collagen (19). By using synthetic triple helical collagen-related peptides, the VWF-binding site has been localized to residues 541-558 of the ␣1CB4(III) fragment of collagen type III (29). Recently, we identified the collagen binding site by cocrystallization of the A3-domain with an inhibitory anti-A3 antibody, RU5, which was confirmed by showing that especially an H1023A mutant abolished binding of VWF to collagen (30). This study was further extended by the analysis of a series of 27 VWF-A3 mutants, which defined the collagen binding site of the VWF-A3-domain to the "front" face of the domain (31), an observation confirmed by Nishida et al. (32).We raised a monoclonal antibody (mAb), 82D6A3, against human VWF that prevents the binding of VWF to collagen (24) and that is antithrombotic in a baboon arterial thrombosis model (33). Since a previous effort to determine the epitope of 82D6A3 using phage display, was not successful (34, 35), we repeated this study us...
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