Recombinant Thyroid Peroxidase-Specific Fab Converted to Immunoglobulin G (IgG) Molecules: Evidence for Thyroid Cell Damage by IgG1, but Not IgG4, Autoantibodies

Abstract

A recombinant autoantibody Fab (SP1.4) to thyroid peroxidase (TPO), cloned from intrathyroidal B cell immunoglobulin genes, interacts with an epitope on TPO recognized by all patients with autoimmune thyroid disease. To compare the biological properties of IgG1 and IgG4 TPO autoantibodies, we converted Fab SP1.4 to full-length immunoglobulins. The SP1.4 heavy and κ light chain variable region genes, spliced by overlap PCR to a mammalian signal peptide, were transferred to expression vectors for human IgG1, IgG4, and κ L chains. Plasmids containing the IgG1 (or IgG4) heavy chain and the κ L chain were cotransfected into SP2/0 mouse myeloma cells. Cells secreting TPO autoantibodies were cloned, and IgG1-SP and IgG4-SP were affinity purified from medium using protein G. Their subclass specificities were confirmed by enzyme-linked immunosorbent assay and fluorometry after binding to Chinese hamster ovary cells expressing cell surface TPO. Further confirmation of SP1.4 Fab conversion to full-length molecules was the ability of protein A to precipitate IgG1-SP and IgG4-SP complexed to [¹²⁵I]TPO. IgG1-SP1.4, IgG4-SP1.4, and Fab SP1.4 had similar high affinities for TPO (K_d = ∼2× 10⁻¹⁰ mol/L). Complexes of [¹²⁵I]TPO and IgG1-SP (but not IgG4-SP) bound to peripheral blood mononuclear cells (PBMC), but not to a B cell line. Flow cytometry demonstrated Fc receptors FcγRI, FcγRII, and FcγRIII on PBMC, but only FcγRII on the B cell line. Together, these data indicate that IgG1-SP/TPO complexes bind to either FcγRI on monocytes or RIII on natural killer cells. In assays for antibody-dependent cytotoxicity using PBMC, ⁵¹Cr release was higher for thyroid cells preincubated with IgG1-SP (13.4%) than with IgG4-SP (2.5%) or with culture medium alone (−0.7%). No specific ⁵¹Cr release was observed when either fibroblasts or Chinese hamster ovary cells expressing cell surface TPO were used as target cells. In conclusion, a human TPO-specific Fab converted to IgG1, but not IgG4, can mediate cytotoxic effects on human thyroid cells in vitro. These observations support the clinical relevance of TPO autoantibody subclass distribution and emphasize the likelihood that, as opposed to being simple markers of thyroid damage, TPO autoantibodies may play a role in the induction of thyroid dysfunction in vivo.