As one of the group’s resident nanobody enthusiasts, on the OPIG retreat this year I presented a talk on shark VNARs, their therapeutic potential and how they differ from VHHs. Here are some of the main points covered:
- Convergent evolution in camelids and cartilaginous fish (i.e. sharks and the like) has led to the existence of heavy-chain antibodies, Y-shaped proteins with no light-chain pairing, in addition to the paired antibodies we all know and love. In sharks, these heavy-chain only antibodies are known as IgNARs. VHHs and VNARs are the antigen-binding fragments or variable domains of these HCAbs/IgNARs, known as nanobodies.
2. The structures of VHHs and VNARs are comparable but differ in a few ways:
- VNARs only have 2 CDR loops compared to the 3 on a VHH (they lose the equivalent of the CDR2 in favour of other hypervariable regions, HV2 and HV4 that connect CDR1 and CDR3)
- Given this lack of CDR2, VNARs are (slightly) even smaller than VHHs in terms of molecular weight, at around ~12kDa compared to VHHs at ~15kDa (Pothin et al., 2020, https://doi.org/10.3390%2Fpharmaceutics12100937).
- Although VHHs are homologous to the VH domain, VNARs are more structurally similar to the VL domain of IgG antibodies (Fernández-Quintero et al., 2022, https://doi.org/10.3389%2Ffimmu.2022.953917)
3. While VHHs are further ahead than VNARs in terms of their clinical success (for reasons detailed in this review from Rossotti et al. https://doi.org/10.1111/febs.15809), there is plenty of interesting research into the potential advantages of VNARs as therapeutics or diagnostic agents:
- Firstly, the larger evolutionary separation between humans and sharks might provide a means to overcome the immune tolerance that can occur when more closely-related animals e.g. mice, are immunized in attempts to raise antibodies against conserved mammalian proteins (Barelle & Porter, 2015 https://doi.org/10.3390/antib4030240).
- Additionally, the high urea concentration of shark blood serum means that VNARs can function in harsh environments, meaning they are very stable (Kovaleva et al., 2014, https://doi.org/10.1517/14712598.2014.937701). Their high thermostability has been suggested to be useful in overcoming cold-chain limitations in warmer climates, which could be used in for example diagnostics for malaria, or in treatments against Ebola (both of which VNARs have been investigated for) (Matz & Dooley, 2019, https://doi.org/10.1016/j.dci.2018.09.007).
In summary, VHHs and VNARs are similar in origin and function. We might expect that the types of epitopes they target could be similar, though it would be interesting to see how the differences in CDRs affects their binding activity. More progress has been made engineering VHHs for therapeutic use, but research shows that VNARs have clear potential. Perhaps the biggest challenge is how we can humanize VNARs to overcome the increased risk of immunogenicity, given the greater genetic distance.
