Thyroid peroxidase autoantibodies obtained from random single chain Fv libraries contain the same heavy/light chain combinations as occur in vivo

Three combinatorial libraries were constructed from unpurified, CD19(+), an d antithyroid peroxidase (anti-TPO) B cells extracted from thyroid tissue o f Graves' disease patients. Fifteen of the 41 randomly derived anti-TPO sin gle chain variable region fragments (scFvs), showed VH1-3/V lambda1-51 or V H1-69/V lambda1-40 heavy/light chain pairing similar to that obtained with TPO-specific scFv derived from an in-cell library. One VH1-3/V lambda1-51 s cFv, A16, showed exactly the same nucleotide sequence as in-cell scFv ICB7, demonstrating that in vivo rearrangement can be obtained from a random com binatorial library. The majority of the scFvs used a heavy chain gene deriv ed from the VH1-3 gene segment, whereas the light chain gene segments used were more heterogeneous, with dominance of the V kappa1-39 and V lambda1-51 gene segments. The anti-TPO scFvs showed high affinities to TPO, with valu es between 0.77 and 12.3 nm, and defined seven antigenic regions on the TPO molecule. The anti-TPO fragments, particularly VH1-3/V lambda1-51 randomly associated scFv B4, which mimic natural H/L pairing, and VH1-3/V lambda1-4 0 in-cell-derived scFv ICA5, efficiently displaced the TPO binding of serum autoantibodies from 20 Graves' disease patients. Our study directly demons trates that antibodies derived from combinatorial libraries are likely to r epresent in vivo pairing, leading to high affinity antibody fragments mimic king the binding of serum autoantibodies to TPO.