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(+), and antithyroid peroxidase (anti-TPO) B cells extracted from thyroid tissue of Graves' disease patients. Fifteen of the 41 randomly derived anti-TPO single chain variable region fragments (scFvs), showed VH1-3/V lambda1-51 or VH1-69/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 scFv, A16, showed exactly the same nucleotide sequence as in-cell scFv ICB7, demonstrating that in vivo rearrangement can be obtained from a random combinatorial library. The majority of the scFvs used a heavy chain gene derived 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 values 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 lambda1-40 in-cell-derived scFv ICA5, efficiently displaced the TPO binding of serum autoantibodies from 20 Graves' disease patients. Our study directly demonstrates that antibodies derived from combinatorial libraries are likely to represent in vivo pairing, leading to high affinity antibody fragments mimicking the binding of serum autoantibodies to TPO.