Genetic origin of IgG antibodies cloned by phage display and anti-idiotypic panning from three patients with autoimmune thrombocytopenia

Abstract

The beneficial use of intravenous immunoglobulins (IVIG) in certain groups of patients with autoimmune thrombocytopenic purpura (AITP) has been proven. AITP is a severe disease in children with a still unknown etiology. It is not clear how IVIG functions in this and other autoimmune diseases. To analyze and compare patient-derived monoclonal IgG antibodies that are bound by IVIG in an anti-idiotypic manner, the combinatorial antibody phage display system was applied. From three different patients with AITP, a large number of clones specifically reacting with IVIG molecules were enriched. The heavy and light chain variable regions were sequenced and compared with each other and with databases. Many variable regions showed extensive replacement mutations within the complementarity-determining regions, while two were identical to germ-line genes. Our data show that the most frequently used germ-line gene loci of these IVIG binders are identical to those observed for many other autoantibodies. This implicates a specific interaction of IVIG particularly with autoantibodies and B cell receptors derived from germ-line genes that are often used for the generation of autoantibodies.

IVIG-bound IgG and IgM cloned by phage display from a healthy individual reveal the same restricted germ-line gene origin as in autoimmune thrombocytopenia

“…In case of the light chains, all but one were derived from the germ‐line genes 3r9C5/DPL23, DPL16/VL3.1, or 2a2.27A12/DPL11 corresponding to the loci 3r, 3l, and 2a2, respectively. Again, the same light chain germ‐line genes have also been detected most frequently in AITP [14]. However, the light chain origin appears to be of only minor importance for the selection by IVIG, because many Fab‐phages without functional light chains were strongly bound by IVIG, and light chain loci 3l and 2a2 were also observed among 3/8 unpanned clones.…”

“…Our previous studies suggested that IVIG bound in an anti‐idiotypic manner to the Fab molecules [14,15]. The method of biopanning with IVIG apparently did not select for original heavy and light chain pairing [14]. Instead, high affinity of IVIG molecules for either antibody chain seemed to result in enrichment of particular Fab‐phages.…”

“…Sequencing revealed that the most frequently used germ‐line gene loci of these IVIG‐selected Fabs were identical to those reported for many other autoantibodies highly specific for the corresponding autoantigen such as dsDNA, acetylcholine receptor, thyroid peroxidase, blood group antigens and others. These loci were 3–23 and 3–30 or 3–30/3–30.5 in case of the heavy chains and 3l, 2a2, and 3r for the light chains [14]. Many of the IVIG‐selected Fabs bound strongly to platelets, in contrast to IVIG‐bound Fabs derived from a healthy individual [15].…”

Some unsettled questions in childhood thrombocytopenia caused by immunologic platelet destruction (acute and chronic ITP)

“…Maybe the observations mentioned above by Kuwana et al offer a unifying concept for the immunopathological processes. Our own results concerning the idiotypic antibody network in ITP showed the key role of anti‐idiotype antibodies (Ab 2) for the downregulation of pathological antibody production 2,3).…”

Molecular Characterization of the Anti-Idiotypic Immune Response of a Relapse-Free Neuroblastoma Patient following Antibody Therapy: A Possible Vaccine against Tumors of Neuroectodermal Origin?

“…Sequencing was done in an automatic sequencer (LI-COR) using the cycle sequencing kit (Amersham Biosciences) and infrared fluorophore (IRD41)-labeled primers. The infrared dye-labeled sequencing primers were PelB and SeqGb for the H chains and OmpA and SeqLb or SeqKb for the L chains (57,63,64 …”