A Llama Nanobody VHH format with beta-cell IC2 autoantibody specificity created by an anti-idiotypic immunization.

The BB-rat monoclonal autoantibody IC2 and its beta-cell surface specificity discovered in the 1980th brought us the explore this unique autoantibody further by DNA cloning and sequencing (Genbank KP709022.1, KP709023.1) and to elucidate further its epitope specificity. The target seems to be certain cell surface CD1d-glycoconjugates (ceramides/sulfatides) also known to be the target for the immune tolerance regulatory NKT-cells. Furthermore, NKT-cells are know the be severely deficient in autoimmune diabetes and other autoimmune diseases. Whether IC2 take part in the etiology and/or pathogenesis of T1D is still unknown.
A series of syngeneic BB-rat monoclonal anti-idiotypic antibodies were raised, of which one particular, named NOVA (Genbank KU324457-KU324458), got our interest, since it could block for the IC2 induced complete inhibition of NKT-cell activation and cytokine release. An anti-anti-idiotypic antibody with IC2-like specificity was isolated from the same fusion indicating the presence of an idiotypic network in the diabetic prone BB-rat.
Aiming to create Nanobody formats with IC2 like beta-cell specificity, we immunized Llamas with the anti-idiotypic NOVA antibody, and succeedingly DNA cloned their VHH regions, to study eventual CDR similarity with the original IC2 autoantibody VH-region. The Llama VHH single strain antibodies showed both beta-cell binding and a strong selective NOVA anti-idiotypic binding. No IC2-VH sequence similarity were found. Unfortunately, neither the original IC2 autoantibody nor the new Nanobody IC2 format had sufficient explicit specificity to beta-cells to be useful for in-vivo noninvasive beta-cell imaging.
Conclusively, the autoantibody IC2 and Llama Nanobody VHH showed in flow cytometry binding both to liver Kuppfer cells expressing CD1d-glycoconjugates, and to a lesser degree to monocytes and macrophages also expressing cell surface CD1d-glycoconjugates. Selected anti-idiotypic antibodies seems stimulatory to NKT-cell proliferation in vitro, with an in-vivo transient anti-diabetogenic effect lowering blood glucose and delaying onset of diabetes in both diabetes prone BB-rats and NOD mice.