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Human Anti-Thyroid Peroxidase Single-Chain Fragment Variable of Ig Isolated from a Combinatorial Library Assembled In-Cell: Insights into the In Vivo Situation¹

Nicolas Chapal^*, Sylvie Peraldi-Roux^*, Damien Bresson^*, Martine Pugniere^*, Jean-Claude Mani^*, Claude Granier^*, Line Baldet, Bernard Guerrier, Bernard Pau^* and Majida Bouanani^2,*

^* Faculté de Pharmacie, Unité Mixte de Recherche 9921, Institut de Biotechnologie en Immunoanalyse et Pharmacologie, Montpellier, France; Service d’Endocrinologie, Centre Hospitalier Universitaire Lapeyronnie, Montpellier, France; and Service d’Oto-Rhino-Laryngologie et de Chirurgie Cervico-Faciale, Guy de Chauliac, Montpellier, France

In an attempt to explore the natural variable heavy and light chain (V_H/V_L) pairing of autoantibodies involved in Graves’ disease, we constructed a phage-displayed Ab library obtained by in-cell PCR of thyroid-infiltrating cells. We report here the molecular cloning and characterization of human single-chain fragment variable regions (scFv) specific for thyroid peroxidase (TPO) generated from this library. On the basis of the nucleotide sequences, three different scFvs were obtained (ICA1, ICB7, and ICA5). All were encoded by genes derived from the VH1 and V1 gene families. Using BIACORE for epitope mapping and kinetic analysis, we showed that these scFvs exhibited high affinity (K_d = 1 nM) for TPO and recognized three different epitopes. The biological relevance of these scFvs as compared with serum anti-TPO autoantibodies was assessed by competition studies. Sera from all the 29 Graves’ disease patients tested were able to strongly inhibit (60–100%) the binding of the 3 scFvs to TPO. These data demonstrate that the in-cell PCR library generated human anti-TPO scFvs that retained the V_H/V_L pairing found in vivo and that the different epitope specificities defined by these scFvs overlapped with those found in the sera of patients with autoimmune thyroid disease.

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