In a recent study from Case Western Reserve University, Matthews et al. developed a laboratory-scale production process to better evaluate fusion proteins that can neutralize emergent severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants using an Azure 600 to evaluate fusion protein affinity for spike protein variants. It is the only system that offers two channel laser based IR detection, chemiluminescent detection with the speed and sensitivity of film, and the ability to image visible fluorescent dyes, standard EtBr and protein gels.
The initial countermeasures against SARS-CoV-2 infection included vaccines, small molecule drugs, and neutralizing monoclonal antibodies. Since then, SARS-CoV-2 variants have emerged that could evade these initial countermeasures. Fusion proteins have been suggested as an ideal alternative that is not affected by variants. This work can help with fusion protein design and improving purification strategies to better study potential fusion protein treatment options.
Introduction to SARS-CoV-2, spike protein, ACE2, and treatment options
The spike (S) protein of SARS-CoV-2 infects human cells by binding to angiotensin enzyme 2 (ACE2)-expressing host cells. This leads to activation of the S protein and membrane fusion as the virus enters the cell. The ability of the S protein to bind to ACE2 is key for the virus’s pathology. The receptor binding domain (RBD) of the S protein is involved in the overall infectivity, immune evasion, and resulting transmissibility of SARS-CoV-2. Due to the high affinity of the RBD of the S protein for ACE2, a possible strategy for counteracting the virus is to use soluble ACE2 as a decoy. Ideally, ACE2 would compete for the S protein binding and thus sequester the virus to prevent it from entering cells.
In vitro system for ACE2 fusion proteins
Previous research has that ACE2 fused with an IgG Fc region (ACE2-Fc) works best at sequestering the SARS-CoV-2 virus, but further characterization is still needed, such as determining the binding affinity for each SARS-CoV-2 variant.
Having an in vitro system to create and test ACE2-Fc proteins against SARS-CoV-2 is important to determine the best countermeasures against current and emerging variants. Matthews et. Al. (1) approached this by generating a cell line that stably expresses ACE2-Fc. The research team qualified the process for obtaining purified ACE2 fusion proteins, and created a system that allows researchers to test a hypothesis: can emerging variants that evade current countermeasures be neutralized by ACE2 fusion proteins?
Creating a cell line to stably express ACE2 fusion proteins
ACE2 is part of the renin/angiotensin system, where angiotensin II regulates the cardiovascular system. ACE2 is shed into the plasma in its catalytically active form to regulate angiotensin II (2). The researchers engineered a mutant ACE2-Fc that would reduce potential effects of having an elevation of ACE2 when used as a decoy fusion protein. To abolish the enzymatic activity while retaining the binding capability to SARS-CoV-2, they mutated two residues (ACE2-(NN)-Fc).
In order to assess the binding affinity of these ACE2 fusion proteins with the S protein, researchers created a Chinese Hamster Ovary (CHO) cell line that stably expressed both ACE2-Fc and ACE2(NN)-Fc. They then confirmed the production of ACE2 fusion proteins using immunoblotting on the Azure 600 (Figure 1). Once confirmed, a laboratory-scaled protein production was performed. After purifying these recombinant proteins, Matthews et al. evaluated their stability under thermal stress, used mass spectrometry to look at their N-glycan profiles, and examined binding activity to the S protein and the different variants.
Characterizing the ACE2 fusion proteins
Protein stability is important for patient safety and must be maintained during the manufacturing process. Matthews et al. evaluated protein stability following different thermal-stress conditions and did not find any changes in stability at 4°C or 25°C after 1 day; however, on day 7 at 25°C, the protein was barely detectable. It is important to note the sensitivity of the ACE2-Fc proteins to thermal-induced stress discovered as this information is important for future storage and handling protocols.
Another important characterization of these fusion proteins was the binding affinity for the spike protein variants. The results found demonstrated that both ACE2-Fc and ACE2(NN)-Fc fusion proteins are active in binding to S proteins. Interestingly, they have greater affinity for alpha, beta and delta S protein variants compared to the parental and omicron variants.
Though a clinical model for evaluating ACE2-Fc proteins as a therapeutic against SARS-CoV-2 does not currently exist, this study provides data supporting this option in the future. Matthews et al have provided data that could aid in the development of ACE2-Fc fusion proteins as a potential countermeasure against SARS-CoV-2. Furthermore, the information found can be used to support new ACE2 fusion protein designs, purification methods, and formulation studies.
Used by Case Western University
- Matthews AM, Biel TG, Ortega-Rodriguez U, Falkowski VM, Bush X, Faison T, et al. (2022) SARS-CoV-2 spike protein variant binding affinity to an angiotensin-converting enzyme 2 fusion glycoproteins. PLoS ONE 17(12): e0278294. https://doi.org/10.1371/journal.pone.0278294
- Turner AJ. ACE2 Cell Biology, Regulation, and Physiological Functions. The Protective Arm of the Renin Angiotensin System (RAS). 2015:185–9. doi: 10.1016/B978-0-12-801364-9.00025-0. Epub 2015 Apr 24. PMCID: PMC7149539. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149539/