The most common organ-specific autoimmune disease in humans involves the thyroid. Autoantibodies against thyroid peroxidase (TPO) are present in the sera of virtually all patients with active disease. We report the molecular cloning of the genes for 30 high-affinity, IgG-class human autoantibodies to TPO from thyroid-infiltrating B cells. Analysis of the putative germline genes used for the TPO human autoantibodies suggests the use of only five different H and L chain combinations involving four H chains and three L chains. In addition, the same combination of H and L chains was found in multiple patients. The F(ab) proteins expressed by these genes define two major, closely associated domains (A and B) in an immunodominant region on TPO. These A and B domains contain the binding sites of 80% of IgG-class TPO autoantibodies in the sera of patients with autoimmune thyroid disease. The present information permits analysis, not previously possible, of the relationship between autoantibody H and L chain genes and the antigenic domains on an autoantigen. Our data, obtained using target organ-derived autoantibodies, indicate that there is restriction in H and L chain usage in relation to the interaction with specific antigenic domains in human, organ-specific autoimmune disease. (J. Clin. Invest. 1993. 92:62-74.)

To characterize the nature ofthyroid peroxidase (TPO) autoantibodies present in the sera of patients with autoimmune thyroid disease, we cloned three IgGl /kappa Fab fragments which bind 125I-TPO. This was accomplished by the molecular cloning and expression in bacteria of IgG gene fragments from B cells infiltrating the thyroid of a patient with Graves' disease. The three Fab fragments (SP2, SP4, and SP5) are coded for by a common heavy chain (VH1, D, JH3) and three related, but different, light chains (VK1, JK2). The SP Fab fragments bind specifically to TPO with high affinities (6 x 10-l"-2 X 10`' M) comparable to those of serum TPO autoantibodies. TPO autoantibodies represented by the SP Fab fragments are present in all 11 patients studied, constitute a high proportion (36-72%) of serum TPO autoantibodies in individual patients and interact with a conformational epitope on TPO. (J. Clin. Invest. 1992. 90:720-726.)

The muscle weakness in myasthenia gravis (MG) is caused by heterogeneous high-affinity IgG autoantibodies to the nicotinic acetylcholine receptor (AChR), a complex ion channel glycoprotein. These antibodies are clearly responsible for reducing AChR numbers at the neuromuscular junction in myasthenia; however, the origins, diversity, specificity and pathogenicity of individual antibodies have not yet been established. We have cloned and characterized four different AChR-specific Fab from an MG patient's thymus by screening an IgG1/kappa gene combinatorial lambda phage library with soluble human AChR labeled with [125I] alpha-bungarotoxin. Unlike most previously cloned human antibodies, all four Fab immunoprecipitated soluble human muscle AChR. Two Fab strongly inhibited binding of mAb to the main immunogenic region on the alpha subunits and one Fab bound to an epitope on the fetal-specific gamma subunit. In sensitivity and fine specificity, these Fab resembled the anti-AChR antibodies found in many MG patients, including the donor. The closest germline counterparts for their heavy chains were in VH families 1, 3 and 4; however, there were many differences consistent with an antigen-driven response of diverse B cell clones. The combinatorial approach holds promise for further analysis of human autoantibodies.

We performed studies to determine whether the binding sites on thyroid peroxidase (TPO) of immunoglobulin antigen binding fragments (Fabs) representing more than 80% of the human autoantibody repertoire overlap with the binding site of monoclonal antibody (Mab) 47, the only Mab whose partial epitope has been defined at the amino acid level (residues 713-721). We also investigated whether these Fabs preferentially recognize native or denatured TPO. None of the Fabs, when bound to radiolabeled TPO, interfered with the ability of Mab 47 to bind to this material. In enzyme-linked immunosorbent assay experiments, the binding of TPO autoantibody Fabs SP1.5, WR1.7, TR1.8, and TR1.9 was greatly diminished by denaturation of TPO. In contrast, binding of Mab 47 was higher to denatured TPO than to intact TPO. Our studies indicate that the Mab 47/C21 epitope lies outside the immunodominant region on TPO. Further, the data confirm that the majority of epitopes for TPO autoantibodies are highly conformational (dependent on the three-dimensional structure of the native protein). Native TPO will be needed to complete the mapping of the epitopes for TPO autoantibodies as well as to determine the amino acids at the autoantibody-antigen-binding sites.

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