Hi everyone,

In this blog post I would like to review an unusual antibody scaffold that can potentially give rise to a new avenue in antibody engineering. Here, I will discuss a couple of papers that complement each others research.

My DPhil is centered on antibody NGS (Ig-seq) data analysis. I always map an antibody sequence to its structure as the three-dimensional antibody configuration dictates its function, the piece of information that cannot be obtained from just the nucleotide or amino acid sequence. When I work with human Ig-seq data, I bear in mind that antibodies are composed of two pairs of light and heavy chains that tune the antibody towards its cognate antigen. In the light of recent research discoveries, Tan et al., found that antibody repertoires of people that live in malaria endemic regions have adopted a unusual property to defend the body from the pathogen (1). Several studies followed up on this discovery to further dissect the yet uncharacterized property of antibodies.

Malaria parasites in the erythrocytic stage produce RIFIN proteins that are displayed on the surface of the erythrocytes. The main function of RIFINs is to bind to the LAIR1 receptors that are found on the surface on the immune cells. The LAIR1 receptor is inhibitory, which leads to inhibition of the immune system. The endogenous ligand of the LAIR1 receptor is collagen, which is found on the surface of body cells. This is to make sure that the immune cells will not be activated against its own body. Activating the LAIR1 receptors is one of the escape mechanisms that the malaria parasite has evolved.

Tan et al., (1) showed that in an evolutionary arms race between human and malaria, our immune system has harnessed the property of RIFINs to bind to LAIR1 against the parasite itself. By doing single B cell isolation and sequencing, it was discovered that antibodies, which are the effector molecules of our immune system, can incorporate the LAIR1 protein in its structure. Taking into account our knowledge of antibody engineering, the idea of incorporating a 100 amino acid long protein into antibody structure is very hard to comprehend. Sequences of these antibodies showed that the LAIR1 insertion was introduced to CDR-H3. Recently, the crystal structure of this construct has become available (2). The crystal structure revealed that the LAIR1 insertion indeed is structurally functional. All 5 of antibody canonical CDRs interact with the LAIR1 protein and its linkers to accommodate the insertion. The CDR-L3 forms two disulfide bonds with the liker to orientate the LAIR1 protein in the way, it will interact with RIFINs. It is worth to stress that LAIR1 sequence differs from the wild type, but the structure is very similar (<0.5 RMSD). The change in sequence and structure is crucial to prevent the LAIR1 containing antibody from interacting with collagen, but only with RIFINs.

Pieper et al., (3) tried to interrogate the modality of LAIR1 insertions into antibody structures. It was performed by single cell sequences as well as NGS of the antibody shift region. It turns out that human antibodies can accommodate two types of insertion modalities and can form   camelid-like antibodies. The insertion of LAIR1 can happen to CDR-H3, leading to the loss of antibody binding to its cognate antigen. Another modality is the incorporation of the LAIR1 protein to the shift region of the antibody. This kind of insertion does not interfere with the Fv domain binding properties, which leads to creating of  bi-specific antibodies. The last finding was the insertion of the LAIR1 into antibody structure where D, J and most of V genes, and the light chain were deleted. The resultant scaffold is structurally viable and only possesses the heavy chain. Hence, it is the evidence that human antibodies can also form camelid-like antibodies. Interestingly, these insertions into the shift region are not exclusive to people that live in malaria endemic regions. By doing NGS of the shift domain from European donors, around 1 in 1000 antibody sequences had an insertion of varying lengths. These insertions are introduced from different chromosomes of both intergenic and genic regions.

To sum up, it is very intriguing that our immune system has evolved to create camelid-like and bi-specific antibodies. It will be very informative to try to crystallize these structures to see how these antibodies accommodate the insertion of LAIR1. Current antibody NGS data analysis primarily concentrates on the heavy chain due to sequencing technology limitations. It will be invaluable information if we could sequence the entire heavy chain as well as adjacent shift region to see how our immune system matures and activates against pathogens.

1. Tan J, Pieper K, Piccoli L, Abdi A, Foglierini M, Geiger R, Maria Tully C, Jarrossay D, Maina Ndungu F, Wambua J, et al. A LAIR1 insertion generates broadly reactive antibodies against malaria variant antigens. Nature (2016) 529:105–109. doi:10.1038/nature16450
2. Hsieh FL, Higgins MK. The structure of a LAIR1-containing human antibody reveals a novel mechanism of antigen recognition. Elife (2017) 6: doi:10.7554/eLife.27311
3. Pieper K, Tan J, Piccoli L, Foglierini M, Barbieri S, Chen Y, Silacci-Fregni C, Wolf T, Jarrossay D, Anderle M, et al. Public antibodies to malaria antigens generated by two LAIR1 insertion modalities. Nature (2017) 548:597–601. doi:10.1038/nature23670