Modulation of the biological activity of thyrotrophin by anti-human thyrotrophin monoclonal antibodies

Abstract

The mechanism by which monoclonal antibodies enhance the biological activity of a number of hormones is poorly understood. One such antibody (GC73), which binds to human but not bovine TSH, enhances the bioactivity of human TSH in vivo. We have investigated whether GC73 enhancement of TSH bioactivity involves potentiation of hormone-receptor activation assessed by the cyclic AMP (cAMP) responses of both primary human thyrocyte cultures and a TSH-responsive human thyrocyte cell line (SGHTL-45). GC73 had no effect on basal cAMP production. In contrast to its enhancement of the bioactivity of human TSH in vivo, it markedly inhibited the cAMP response to 1 and 10 mU human TSH/ml in primary thyrocytes. This effect was dose-dependent with neutralization of the bioactivity of TSH occurring at 2 mg GC73/ml. GC73 had no effect on the bioactivity of bovine TSH. In contrast, a second anti-TSH monoclonal antibody (TC12), which binds to both human and bovine TSH, inhibited the bioactivity of both species of TSH. Similar results were obtained using SGHTL-45 cells, although the peak concentrations of cAMP were lower. We conclude that binding of GC73 to human TSH resulted in inhibition rather than enhancement of the in-vitro biological activity of human TSH. We suggest that GC73 enhancement of human TSH bioactivity seen in vivo does not result from a mechanism involving potentiation of receptor activation by human TSH.

A functional site on the human TSH receptor: a potential therapeutic target in Graves’ disease

“…1). Furthermore, the fact that we obtained five independent hybridomas against residues 381–384 following immunization with the whole N‐terminal extracellular region (Shepherd et al ., 1999) and that two other groups have also generated antibodies against this epitope using different immunization protocols (Costagliola et al ., 2002b; Jeffreys et al ., 2002) is intriguing, illustrating the immunogenicity of this site.…”

“…The culture medium was removed from FLD4 cells in 24‐well plates (approximately 10 5 cells/well) and replaced with ‘hypotonic Hepes’ (final volume 200 µl) containing bovine TSH (usually 100 µU/ml) or human sera (usually 10% by volume; 3 mU/ml for LATS‐B standard) in the presence or absence of purified monoclonal antibody (up to 40 µg/ml). After incubation for 2 h at 37°C, the supernatants were removed and their cAMP content measured by radioimmunoassay (Page et al ., 1990).…”

The effect of basic fibroblast growth factor on the growth and function of human thyrocytes

“…Tissue culture SGHTL-189 cells were prepared and maintained in log-phase tissue culture as described for other SGHTL-type cells (Page et al 1990). For growth and cyclic AMP (cAMP) accumulation experiments, cells were subcultured in 24-well multiwell plates at an initial density of 4-7 IO4 cells per well.…”

Thyrocyte release of asymmetric dimethylarginine does not account for human thyrocyte inhibition of endothelial cell cyclic GMP

“…Primary cultures of human thyrocytes were prepared by dispase type II digestion of surgical specimens from patients with multinodular goitre or Graves' disease, as previously described (13). Primary thyrocytes were maintained in monolayer culture in Ham's F10 medium containing 5% (v/v) newborn calf serum, 10 mg/ml insulin, 10 ng/ml somatostatin, 10 ng/ml glycine-histidine-lysine acetate, 3.6 ng/ml hydrocortisone, and 5 mg/ml transferrin (5H medium) (14).…”