Antibody diversification by templated insertions: impact, mechanism and exploitation
This project aims at investigating and exploiting a new mechanism of antibody diversification that we recently discovered (Tan et al., 2015). Briefly, we found that up to 10% of malaria-exposed individuals make antibodies in which the DNA encoding the extracellular domain of LAIR1 (a collagen-binding inhibitory receptor encoded on chromosome 19) is inserted in the immunoglobulin (Ig) genes, either between the V and DJ segments or in the switch region. The inserted LAIR1 exon undergoes somatic mutations that abolishes collagen binding and leads to the generation of broadly reactivity antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes. These surprising findings raise several questions as to the mechanism of templated insertions and their contribution to antibody diversity which will be addressed in the current project. The first aim is to develop new sequencing strategies to identify templated insertions in Ig genes, to establish their frequency and origin. The second aim is to understand the mechanisms that cause templated insertions in Ig genes. The studies will be performed using naïve B cells undergoing isotype switching in vitro and by manipulating the factors that control non-homologous end joining (NHEJ) and by offering different RNA or DNA substrates for insertion. The third aim is to produce through protein engineering a new format of bispecific antibodies by inserting antigen-binding domains, such as VHH, scFv or viral receptors, in the antibody VH-CH1 elbow, a format that we have recently identified in LAIR1-containing antibodies. The fourth aim is to engineer primary B cells to make bispecific antibodies by inserting selected domains in the switch region leading to their expression in the VH-CH1 elbow. As a proof of concept, we will insert domains characterized in Aim 3. The project proposed is strongly supported by preliminary data, addresses a fundamental question with regard to the mechanisms of antibody diversification and will explore a new approach to engineer human B cells for adoptive transfer that could be used to protect against a variety of pathogens.