Characteristics of a Human Monoclonal Autoantibody to the Thyrotropin Receptor: Sequence Structure and Function

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.

Relevance of TSH-receptor antibody levels in predicting disease course in Graves’ orbitopathy: comparison of the third-generation TBII assay and Mc4-TSI bioassay

“…[6][7][8] Classical TBII assays are limited by their inability to discriminate between thyroid-stimulating antibodies (TSAbs) and thyroid-blocking antibodies (TBAbs). However, the most newly developed thirdgeneration TBII assay inhibits binding of a labeled TSAb (monoclonal Ab clone #M22) rather than labeled TSH to the TSH receptor 9,10 This results in enhanced sensitivity and specificity vs earlier assays using radiolabeled TSH. 6,8,11,12 The TSI bioassay measures cyclic adenosine monophosphate production after TSAb binds to TSH receptor.…”

Analysis of the interaction between human steroid 21-hydroxylase and various monoclonal antibodies using comparative structural modelling

“…A recombinant Fab of M21-OH5 was produced in Escherichia coli using a previously described method (10). The HC and the LC RT-PCR products were cloned into Immunozap H/L vector (Stratagene Europe, Amsterdam, The Netherlands) under the lacZ promoter and the cloning confirmed by sequencing.…”

“…The HC and the LC RT-PCR products were cloned into Immunozap H/L vector (Stratagene Europe, Amsterdam, The Netherlands) under the lacZ promoter and the cloning confirmed by sequencing. M21-OH5 Fab was expressed in HB2151 cells (Amersham Biosciences) using conditions as described previously (10). M21-OH5 Fab preparations contained in the bacterial cell extract in PBS with 1% Triton X-100 and 1 mmol/l phenylmethylsulphonylfluoride (PMSF) were analysed for 21-OH binding activity as described before (6).…”

The Genetics of the Thyroid Stimulating Hormone Receptor: History and Relevance

“…3). Through a series of sequence/structure alignments investigators first modeled the leucine-rich domain, the cleavage domain (CD), and the TMD of the TSHR (13,14); recently, the crystal structure of the TSHR ectodomain in complex with a TSHR antibody Fab region has been determined (15). Activation of the receptor involves binding of cognate hormone to the large ectodomain of the TSHR likely followed by interactions between receptor TMDs and G proteins.…”