Year of the Camelid: Antibody Style

The UN General Assembly has declared 2024 the International Year of Camelids. The declaration is intended to raise awareness of the economic and cultural importance of these animals to human populations around the world. Here at Addgene, we love camelids too, though not just for the reasons the UN describes. In honor of the Camelid family, I wanted to take a moment to revisit some of the ways these animals have impacted humans through biomedical research.

The Camelid family is composed of camels, llamas, and alpacas (and their undomesticated counterparts) - not exactly species that jump to mind when we think “biomedical research". The family entered the biomedical field’s spotlight after researchers discovered heavy-chain only antibodies in the serum of a camel and soon after found similar antibodies in llamas and alpacas (Figure 1). At the time, researchers were already on a quest for smaller forms of antibodies and had begun developing Fab fragments and scFvs. But the potential for an even smaller, single-domain antibody fragment was exciting, and it wasn’t long until the field was off and running with camelid-derived single-domain antibodies (sdAb), commercially known as nanobodies (Arbabi-Ghahroudi, 2017)! 

Figure 1: Comparison of a heavy-chain only antibody and single-domain antibody, under the commercial "Nanobody" name. 

We’ve talked about sdAbs a number of times on this blog over the years and have described how they compare to other antibodies and affinity reagents. But for a quick refresher, some of the advantages of sdAbs over conventional antibodies include: 

• Excellent tissue penetration, thanks to their small size of 12–15 kDa
• Ease of cloning and expression from plasmids, due to only needing one open reading frame
• Ability to access unique epitopes with high affinity, due to a greater ability to target concave epitopes
• Suitability for in vivo experiments, due to their simple structure 
    

sdAbs in research

These advantages have contributed to sdAbs becoming valuable tools for diverse applications. For example, RANbodies can be used as an alternative to conventional primary antibodies, and there are unique benefits to using sdAbs as secondary antibodies. sdAbs, however, really shine in vivo, like “chromobodies,” which are single-domain antibodies genetically fused to fluorescent proteins and used to visualize target antigens in living cells or tissues. sdAbs can also be used to manipulate cellular functions, like in this early example we highlighted back in 2015, where sdAbs were used to create GFP scaffolds for transcriptional activation. 

sdAbs in the clinic

sdAbs have been finding their way out of the lab and into the clinic as well. The first sdAb based therapeutic was approved in 2018, with many others in clinical trials (Jin, et al. 2023). In addition to therapeutics, there is a lot of excitement around using sdAbs in diagnostic applications. For example, fluorescently and radioactively labeled sdAbs have become appealing candidates for the detection of different cancers (Jin, et al. 2023). 

So where do all these sdAbs come from? In the early years of sdAbs development, the process started with immunizing a camelid (usually llamas or alpacas). But because sdAbs are so amenable to recombinant expression (and because maintaining a llama facility is out of reach and unappealing to many labs), the field is moving towards using synthetic single-domain antibody libraries to generate new sdAbs. There has also been some interesting work done developing transgenic mice that express camelid variable domains (aka nanomice), which gives researchers the “best of both worlds” - a familiar lab model and a source of sdAbs (Figure 2) (Xu, et al., 2021). 

Figure 2: Genetically modified mice offer an alternative method to producing single-domain antibodies without the need for a species that naturally produces heavy-chain only antibodies. (A) Overview of genetic modifications made to generate the nanomouse. (VHH = variable domains of heavy chain only antibodies) (B) Nanomice express hybrid heavy-chain only antibodies that are the source of mouse-derived single-domain antibodies. Image created in BioRender and adapted from Xu, et al., 2021.

The single-domain antibody field has come a long way since the serendipitous discovery of heavy-chain only antibodies in a camel. And I suspect we will only continue to see their impact grow as new sdAbs become easier to generate and more widely adopted. So thank you to the camelids who inspired this versatile family of tools, the applications they have already enabled, and the discoveries yet to come.   

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References and resources

References

Arbabi-Ghahroudi M (2017) Camelid Single-Domain Antibodies: Historical Perspective and Future Outlook. Front Immunol 8:1589. https://doi.org/10.3389/fimmu.2017.01589

Jin B, Odongo S, Radwanska M, Magez S (2023) Nanobodies: A Review of Generation, Diagnostics and Therapeutics. Int J Mol Sci 24:5994. https://doi.org/10.3390/ijms24065994

Xu J, Xu K, Jung S, et al (2021) Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants. Nature 595:278–282. https://doi.org/10.1038/s41586-021-03676-z 

More resources on the Addgene blog

• Introduction to Antibodies 
• Secondary Nanobody Toolbox
• No Llamas Required - Synthetic Nanobodies Against Membrane Proteins

More resources from addgene.org 

• Plasmids encoding nanobodies at Addgene
• Sybody Generation Toolbox from Addgene