Human monoclonal thyroid stimulating autoantibody.

A monoclonal autoantibody (MAb) with powerful thyroid stimulating activity has been produced from lymphocytes from a patient with Graves' disease. The autoantibody and its Fab fragment bind to the thyroid stimulating hormone (TSH) receptor (TSHR) with high affinity, inhibit labelled TSH binding to the receptor and stimulate cyclic AMP production in Chinese hamster ovary cells transfected with TSHR. TSHR autoantibodies with TSH agonist or antagonist activities from patients' serum samples are effective inhibitors of labelled monoclonal autoantibody binding to TSHR. Thus, the human monoclonal autoantibody has all the characteristics of serum TSHR autoantibodies. Its availability has important implications for new studies on the pathogenesis of Graves' disease.

Characteristics of a monoclonal antibody to the thyrotropin receptor that acts as a powerful thyroid-stimulating autoantibody antagonist.

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.

Development of antibodies that interfere with the collagen-VWF-GPIb axis as new antithrombotics.

The epitope and the antithrombotic effect of 6B4, an antibody that inhibits GPIb, the receptor for von Willebrand Factor (VWF) on blood platelets, and of 82D6A3, an antibody against VWF that prevents the binding of VWF to collagen, were characterised. By using canine-human chimeras, alanine-scans, phage display, mutant analysis and modeling both the epitope of 6B4 in the N-terminal domain of GPIb, and of 82D6A3 in the VWF-A3 domain, could be mapped. As both epitopes furthermore are part of the ligand binding sites, this at once also explained the mechanism of the inhibition by the antibodies. Next both antibodies were tested in a thrombosis model in a stenosed artery in baboons, where they showed potent antithrombotic activities, without a noteworthy prolongation of the bleeding time. With this we thus could reveal two new strategies to prevent arterial thrombosis, which presumably may be safer than the currently available antiplatelet agents.

The collagen-binding leech products rLAPP and calin prevent both von Willebrand factor and alpha2beta1(GPIa/IIa)-I-domain binding to collagen in a different manner.

Calin and rLAPP are two natural inhibitors that are able to inhibit the vWF-binding and platelet adhesion to collagen both under static and flow conditions. In this study we demonstrate that both rLAPP and Calin prevent alpha2I-domain binding to human collagen type I with an IC50 of 5 microg/ml. However, although both vWF and alpha2I-domain binding to collagen is prevented by rLAPP and Calin, the latter two do not bind to the same collagen site since Calin only partially could compete with rLAPP for binding to collagen. Also vWF and the alpha2I-domain were unable to compete completely with each other for the binding to collagen. So the following hypothesis can be made: the binding sites of vWF and of the alpha2I-domain on human collagen type I are different but close to each other since rLAPP could inhibit both interactions, and thus should bind to an overlapping epitope. The Calin preparation on the other hand may still contain two active principles, one interfering with vWF-binding, the other with the alpha2I-domain-binding to collagen.

Selection of phages that inhibit vWF interaction with collagen under both static and flow conditions.

Phages from a pentadecamer phage display library were selected for binding to vWF by affinity panning. Bound phages were selectively eluted with human collagen type I. After the third round of panning 95% of individual phage clones bound to vWF. The B8-phage inhibited the binding of collagen to vWF with an IC50 of 0.6 x 10(10) phages/ml, and of vWF to collagen with an IC50 of 1.0 x 10(10) phages/ml at 0.5 microg/ml vWF. Under flow conditions, 1.5 x 10(11) B8-phage/ml nearly completely inhibited platelet deposition on a human collagen type I coated surface at a shear rate of 1200 s(-1), while phages without an insert had no effect. The peptide corresponding to the one displayed on the B8-phage competed with the phage for binding to vWF with an IC50 of 30 microg/ml (16 microM). The peptide furthermore inhibited vWF-binding to collagen with a maximum of 40% at a concentration of 1.25 mg/ml (650 microM), higher concentrations of peptide could not improve this. We thus have selected phages that are potent vWF-binders and that can be used as tools to detect vWF, to inhibit vWF-collagen interaction and to further analyse the role of vWF-collagen binding.

A monoclonal antibody to platelet type III collagen-binding protein (TIIICBP) binds to blood and vascular cells, and inhibits platelet vessel-wall interactions.

TIIICBP is a new platelet receptor involved in platelet-type III collagen and platelet-subendothelium interactions. This receptor is composed of a doublet of 72-68 kDa proteins. In this study, the major protein (68 kDa) was purified and used to produce monoclonal antibodies. One of these antibodies, 7F4, binds to platelets as confirmed by flow cytometry. 7F4 inhibited platelet contact, spreading and aggregation induced by type III collagen. Under flow conditions, 7F4 prevented platelet interactions with type III collagen, endothelial cell matrix and the KOGEOGPK type II collagen octapeptide: the specific sequence recognized by TIIICBP. On the other hand, 7F4 had no effect on platelet-type I collagen interactions. TIIICBP was also detected on lymphocytes, granulocytes and monocytes. TIIICBP was expressed on endothelial cells and fibroblasts but not on smooth-muscle cells. These results show that TIIICBP is expressed on several cell types and participates in cell adhesion to the subendothelium.

The integrin alpha 2 beta 1 (GPIa/IIa)-I-domain inhibits platelet-collagen interaction.

The integrin alpha 2 beta 1 is a major cellular receptor for collagen. The alpha 2 subunit contains an +/- 200 amino acids inserted domain (I-domain) in the N-terminal region. A certain degree of homology exists between the I-domains found in integrins, collagen and the A-domains of vWF. The alpha 2-I-domain encoding region (aa residues D145 to S334) was obtained by RT-PCR from mRNA of non stimulated human PBL's. The primers were designed to introduce the necessary restriction sites for cloning of the DNA fragment in frame downstream of the malE gene, as well as a stop codon after the last triplet. The resulting construct pMAL-c2-alpha 2-I allows the expression of the I-domain, fused to the C-terminus of maltose binding protein (mal). The alpha 2-I-mal is purified from the bacterial extract by affinity chromatography on an amylose column. The purified alpha 2-I-mal has been characterized by ELISA's. The alpha 2-I-mal bound to immobilised collagen type I in a concentration dependent manner and could be blocked by the functional monoclonal anti-alpha 2 beta 1 antibody 6F1. The interaction of alpha 2-I-mal with collagen furthermore is Mg(2+)-dependent since the binding was inhibited in the presence of 10 mM EDTA or 10 mM Ca2+ but sustained in the presence of 10 mM Mg2+. Finally, alpha 2-I-mal itself was able to inhibit adhesion of washed platelets to collagen immobilised on a microtiterplate in a dose-dependent manner (alpha 2-I-mal IC50:0.7 microM) as well as platelet aggregation induced by collagen type I (alpha 2-I-mal IC50:0.7 microM). With these results we could confirm that the alpha 2-I-domain represents the collagen-binding site of alpha 2 beta 1 and we furthermore could indicate that this domain is able to prevent platelet adhesion to collagen and collagen-induced platelet aggregation, pointing to the primordial role of alpha 2-I-mal and hence of alpha 2 beta 1 in platelet-collagen interaction.

Characteristics of a human monoclonal autoantibody to the thyrotropin receptor: sequence structure and function.

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.

An anti-idiotypic antibody with an internal image of human interferon-gamma and human interferon-gamma-like antiviral activity.

D9D10, a monoclonal antibody that inhibits the biological activity of human interferon-gamma (IFN-gamma), was used to generate monoclonal anti-idiotypic antibodies. After a first selection, the monoclonal anti-idiotypic antibody AA1E5 was chosen to be fully characterized. To the best of our knowledge this is the first description of a monoclonal antibody with an IFN-gamma-like antiviral activity; AA1E5 competed with IFN-gamma for binding to D9D10 indicating its anti-idiotypic character. However, AA1E5 also fully mimics HuIFN-gamma as it not only binds to the HuIFN-gamma-receptor, where it competes with HuIFN-gamma, but more importantly AA1E5 and its Fv fragment, cloned and expressed in Escherichia coli, mimic the antiviral activity of HuIFN-gamma. Indeed, 15 microg of AA1E5 and 2.5 microg of its Fv fragment had an effect comparable to that of 10 IU of HuIFN-gamma in an antiviral assay on A549 cells. Sequence comparison between the complementarity determination regions of the antibody and the sequence of HuIFN-gamma revealed that both the heavy chain variable domain, VH, and the kappa light chain variable domain, Vkappa, have epitopes of 3-4 amino acids that are present in the HuIFN-gamma sequence, some of which contribute to receptor binding, as identified by Walter et al. [M. R. Walter, W. T. Windsor, T. L. Nagabhushan, D. J. Lundell, C. A. Lunn, P. J. Zauodny & S. K. Narula (1995) Nature 376, 230-235].

Identification of peptides, selected by phage display technology, that inhibit von Willebrand factor binding to collagen.

A repeated selection of phages from a cyclic hexapeptide phage display library resulted in an enrichment of phages that bound to the monoclonal antibody (MoAb) 82D6A3 (an anti-von Willebrand Factor [vWF] antibody that inhibits binding of vWF to collagen). Two clones were selected that bound both to MoAb 82D6A3 and to rat tail collagen type I in a specific and dose-dependent manner. The two phage clones were further used in a two-direction competition experiment with vWF. vWF was able to displace phages from collagen in a dose-dependent manner with an IC50 of 35 micrograms/mL and phages were able to inhibit vWF binding to collagen. With the use of specific primers, the sequence of the cysteine-flanked hexapeptide inserts could be deduced. The two phage clones carried an almost identical sequence, CVWLWEQC and CVWLWENC, with a substitution of an N for a Q at position 6 of the hexapeptide. Sequence comparison with the known vWF sequence showed the presence of a comparable sequence at position 1129-1136 (VWTLPDQC), located between the collagen-binding A3-domain and the D4-domain. The two cyclic peptides, the putative corresponding vWF peptide, and a peptide with a scrambled cyclic sequence were synthesized. The two cyclic peptides inhibited vWF binding to rat tail collagen type I in a dose-dependent manner, whereas the linear vWF peptide and the scrambled cyclic peptide were inactive. For half maximal inhibition, 100 +/- 12.7 micromol/L and 34.8 +/- 8.59 micromol/L (mean +/- SEM, n = 3) of the N- and the Q-peptide, respectively, were needed. The two cyclic peptides were also able to inhibit vWF binding to calfskin and human collagen type I, but effective concentrations were some 5 to 10 times higher.

Distinct sequences of the glycoprotein Ib-binding domain of von Willebrand factor involved in shear induced platelet aggregation.

Shear-induced platelet aggregation (SIPA) requires von Willebrand factor (vWF) binding to the platelet receptors GPIb and alphaIIbbeta3. In order to determine the vW F sequences involved in SIPA at 4000/s, we studied the llb 3 effect of three monoclonal antibodies (mabs) 724, 713 and 328 to the A1 domain of vWF. We found that mab 724 induced an enhanced SIPA via a Fc gamma-receptor independent mechanism. In contrast, mab 713 and mab 328 could inhibit SIPA by 52 and 91% , respectively. Based on distinct effects on SIPA, we can propose the following working model for the interaction between vWF and GPIb: mabs 713 and 328, which block SIPA, may recognize an epitope that is involved in binding to GPIb, whereas mab 724, which increases SIPA in the presence of vWF, may mimic the effect of botrocetin when binding to vWF, by inducing an active conformation of vWF, which may be more sensitive to high shear rate.

Platelet aggregation induced by a monoclonal antibody to the A1 domain of von Willebrand factor.

Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of alphaIIb beta3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to alphaIIb beta3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds-1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds-1, DSP was increased from 5.9% +/- 3.5% in the absence of vWF to 32.7% +/- 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to alphaIIb beta3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds-1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to alphaIIbbeta3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab')2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcgamma receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.