Limitations of the semisynthetic library approach for obtaining human monoclonal autoantibodies to the thyrotropin receptor of Graves' disease

Graves' disease (GD) is characterized by the presence of autoantibodies aga inst the TSH-receptor (TSH-R) which are pathogenic and, upon binding to the receptor, trigger intracellular signal transduction. The autoantibodies ar e oligoclonal and as they are responsible for disease activity, their chara cterization would lead to a better understanding of the development of GD. Attempts to isolate anti-TSH-R antibodies from patients have proved to be d ifficult due to the exceedingly low serum levels due to rarity of these B c ells, together with difficulties in obtaining purified TSH-R capable of int eracting with patients autoantibodies. We employed phage antibody display t echnology and performed selection with a previously characterized semisynth etic antibody library on the purified extracellular ectodomain of the TSH-R . We report the isolation of six different anti-TSH-R monoclonal phage anti bodies (moPhabs) from this library. All the moPhabs recognized TSH-R and it s recombinant fragments by Western blotting, but failed to recognize the na tive TSH-R by flow cytometry. Consequently, the moPhabs did not lead to TSH -R activation. As these were the first moPhabs to TSH-R, they were analysed in terms of nucleotide and amino acid sequence and epitope specificity on the receptor. The moPhabs used immunoglobulin VH1 and VH3 germ line genes, all associated with V lambda 3 genes. Interestingly, the CDR3 regions of al l moPhabs were remarkably similar, though not identical. In light of the co mmon CDR3 usage, the epitopes recognized on TSH-R appeared to be restricted to amino acids residues 405-411 and 357-364. In summary, our results show that semisynthetic libraries may be limited in isolating human monoclonal a ntibodies that resemble pathogenic antithyrotropin receptor autoantibodies present in patients with GD. It is likely that until preparations of purifi ed TSH-R that can be recognized by patients autoantibodies become available , similar to the recently described glycosylphosphatidylinositol (GPI) anch ored TSH-R ectodomain, monoclonal antibodies from phage antibody display to TSH-R will be limited for isolating the rare, pathogenic antibodies of GD.