The human leucocyte antigen (HLA)-G, which consists of seven splice variants, is a tolerogenic immune checkpoint molecule. It plays an important role in the protection of the fetus from the maternal immune response by binding to inhibitory receptors, including leukocyte Ig-like receptors (LILRs). Recent studies have also revealed that HLA-G is involved in the progression of cancer cells and the protection from autoimmune diseases. In contrast to its well characterized isoform, HLA-G1, the binding activities of other major HLA-G isoforms, such as HLA-G2, toward available anti-HLA-G antibodies are only partially understood. Here, we investigate the binding specificities of anti-HLA-G antibodies by using surface plasmon resonance. MEM-G9 and G233 showed strong affinities to HLA-G1, with a nM range for their dissociation constants, but did not show affinities to HLA-G2. The disulfide-linker HLA-G1 dimer further exhibited significant avidity effects. On the other hand, 4H84 and MEM-G1, which can be used for the Western blotting of HLA-G isoforms, can bind to native HLA-G2, while MEM-G9 and G233 cannot. These results reveal that HLA-G2 has a partially intrinsically disordered structure. Furthermore, MEM-G1, but not 4H84, competes with the LILRB2 binding of HLA-G2. These results provide novel insight into the functional characterization of HLA-G isoforms and their detection systems.
VHH, the antigen-binding fragment of a heavy chain-only antibody, is a useful component of antibody-based therapeutics. Thermal stability, represented by the melting temperature (Tm), is one of the key factors affecting the developability of antibody-based therapeutics. In this study, we examined whether the in silico free energy score dStability can be used to design mutants with improved Tm compared to the anti-lysozyme VHH, D3-L11. After verifying that exhaustive mutagenesis was inefficient for improving Tm, we performed a two-round rational approach that combined dStability calculations with a small number of experiments. This method improved the Tm by more than 5 °C in several single mutants including A79I. It reduced the affinity for the antigen by less than 1.6-fold. We speculate that stabilization of A79I required exquisite compatibility among neighboring residues to fill in the internal cavity in the protein. Given that we identified only one mutation that could simultaneously improve Tm and almost maintain affinity, we concluded that achieving both is extremely difficult, even with single mutations that are not located in the paratope. Therefore, we recommend using a variety of approaches when trying to achieve such a feat. Our method will be a useful complementary approach to other existing methods.
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