The COVID-19 pandemic has affected us all with many disruptions in daily life. Such a pandemic makes one realize the importance of our body’s natural defense system (i.e. our immune system) to tackle infectious diseases. Beautifully intricate processes coordinate in our body once infected with foreign objects called antigens. One of the main ways our immune system tackles these antigens is by creating antibodies that bind and thus block (or also known as “neutralize”) the antigen’s ability to target host cells. To create neutralizing antibodies, we would either need to be vaccinated with a decoy antigen that is similar to the actual infectious agent or acquire antibodies from natural infection. However, the making of antibodies is a trial and error process with many antibodies not able to actually neutralize foreign antigens. Moreover, antibody levels can wane over time, which is why booster vaccine shots are sometimes necessary. Therefore, the number of neutralizing antibodies at a given time is dynamic and important to predict our protection from future infection. Thus, we set out to create a rapid test to quantify the amount of neutralizing antibodies from a couple drops of blood, allowing users to measure their protection from SARS-CoV-2.

To this end, we utilized the de novo designed LOCKR (Latching, Orthogonal Cage/Key pRotein) system as a biosensor for measuring SARS-CoV-2 components and antibodies¹. The two-state LOCKR system is designed to be switchable, thus ideal for use as a biosensor². LOCKR contains 2 proteins: 1) Cage protein: contains a 5-helical cage domain tethered to and interacting with the 1-helical latch domain, 2): Key protein: contains the 1-helical key domain that also has affinity to the cage domain. To transform LOCKR into a sensor for SARS-CoV-2 components (specifically the receptor binding domain (RBD) from the spike protein), a de novo designed binder (with picomolar affinity) to RBD called LCB1³ was embedded on the end of the latch so that binding of RBD to LCB1 weakens the binding between cage and latch domains, strengthening the binding between cage and key domains, and thus allowing for the 2 LOCKR proteins to associate. To allow for readout of this binding event, split luciferase was added to the LOCKR proteins where the smaller bit was embedded in the latch and the larger portion attached to the end of the key protein. For this RBD sensor, the cage protein is called lucCageRBD and the key protein is called lucKey². Thus, increased amounts of RBD binding to LCB1 in the cage protein translates to increased bioluminescence from the now reconstituted luciferase.

However, this set up is only measuring the concentration and affinity of RBD. To transform this sensor to measure SARS-CoV-2 neutralizing antibodies, we focused on the antibody’s ability to disrupt RBD binding to LCB1⁴. We focused on this binding event as most neutralizing antibodies identified are blocking this interaction. Addition of RBD leads to the sensor displaying increased bioluminescent signal, while addition of antibodies that block RBD interaction with LCB1 in lucCageRBD (a proxy for neutralizing antibodies) leads to decreased bioluminescent signal with complete reversal of signal indicative of complete neutralization. This system is inherently a 4-protein component (quaternary) system compared to previous assays where 3 or less proteins are required⁵. With previous systems, usually the RBD is tagged with some molecular readout, thus new RBD variants for the sensor need to be engineered and verified. This is especially cumbersome for SARS-CoV-2 as there are a panoply of variants. Our quaternary system avoids RBD labeling, allowing for interchangeability of RBD variants, and allows for the unique ability to discern between high amounts of weakly neutralizing antibodies compared to low amounts of strongly neutralizing antibodies, which would be useful to characterize a person’s immunity. Moreover, this platform is generalizable and can be applied to other infectious diseases and antibodies against those infectious agents.

These tools were not possible without advancements in de novo protein design which goes hand-in-hand with developments in protein structure prediction programs. These devices are just a glimpse into what de novo protein design can do and showcases that these proteins are not only useful for therapeutics (LCB1 can neutralize SARS-CoV-2 infection in animal models and is being tested in other contexts) but also for diagnostics. Instead of waiting for evolution to take its time and produce dynamic and useful proteins, we believe that de novo proteins can orders-of-magnitude quicken this process for making designer molecules that tackle age-old problems⁶.

¹ Langan RA, Boyken SE, Ng AH, Samson JA, Dods G, Westbrook AM, et al. De novo design of bioactive protein switches. Nature 572, 205–210 (2019).

² Quijano-Rubio A, Yeh H-W, Park J, Lee H, Langan RA, Boyken SE, et al. De novo design of modular and tunable protein biosensors. Nature doi:10.1038/s41586-021-03258-z (2021).

³ Cao, L. et al. De novo design of picomolar SARS-CoV-2 miniprotein inhibitors. Science 370, 426–431 (2020).

⁴ Zhang, J.Z., Yeh, HW., Walls, A.C. et al. Thermodynamically coupled biosensors for detecting neutralizing antibodies against SARS-CoV-2 variants. Nat Biotechnol https://doi.org/10.1038/s41587-022-01280-8 (2022).

⁵ Tan, C. W. et al. A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2–spike protein–protein interaction. Nat. Biotechnol. 38, 1073–1078 (2020).

⁶ Cao, L., Coventry, B., Goreshnik, I. et al. Design of protein binding proteins from target structure alone. Nature https://doi.org/10.1038/s41586-022-04654-9 (2022).