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

Abstract

In an attempt to explore the natural variable heavy and light chain (VH/VL) 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 Vλ1 gene families. Using BIACORE for epitope mapping and kinetic analysis, we showed that these scFvs exhibited high affinity (Kd = 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 VH/VL 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.

Human recombinant anti-thyroperoxidase autoantibodies: in vitro cytotoxic activity on papillary thyroid cancer expressing TPO

“…These regulatory proteins are expressed on NPA cell membrane and probably partially protect them from CDC. Combining the various human recombinants anti-TPO aAbs we selected by phage display and characterised in term of epitopes (Bresson et al, 2003(Bresson et al, , 2004Chapal et al, 2000;Rebuffat et al, 2006), could make it possible to improve complement activation.…”

“…Using the phage-display technology, we previously selected and characterised human recombinant anti-TPO aAbs (Chapal et al, 2000. These human recombinant anti-TPO aAbs mimic human anti-TPO aAbs present in the sera of patients suffering from AITD and recognise the human TPO (hTPO) immunodominant region (Bresson et al, 2003, 2004, 2005, Rebuffat et al, 2006.…”

Is Chronic Lymphocytic Leukemia a Mistake of Tolerance Mechanisms?

“…This also can explain that unmutated CLL cases and mutated CLL cases express different antibody repertoires and different VH genes (Fais et al 1988;Johnson et al 1997). Current data support that CLL cells are in active (auto) antigen driven receptor editing, presumably by keeping away from autoreactivity associated with preferential autoimmune linked IGHV gene utilization in CLL patients like IGHV3-21, IGHV4-34, IGKV1-17 (Foreman et al 2007;Hadzidimitruiou et al 2009) and also IGHV5-51 and IGHV1-69 in unmutated IgVH genes (Chapal et al 2000;Vanura et al 2008). Interestingly, highly polyreactive antibodies are expressed frequently by unmutated CLL, but only rarely by mutated cases, supporting the view that the receptor editing mechanism is significantly active to try to elude autoimmunity in CLL.…”

Mining human antibody repertoires

“…Addressing this concern, phage display has also been used to mine non-combinatorial antibody libraries, i.e., antibody libraries that retain original heavy and light chain pairings of human antibody repertoires through in-cell RT-PCR. 110 This method is of particular interest for the identification of human antibodies in autoimmune repertoires.…”