Mutagenesis of ACE2 for Enhanced Affinity
Soluble decoy receptors should ideally bind the SARS-CoV-2 spike tighter than, and out-compete, the wild type (WT) ACE2 receptor. Tight affinity is also necessary for effective virus neutralization at typical doses for biologic drugs. The importance of affinity is further emphasized when soluble decoys are considered against the therapeutic alternative of affinity-matured monoclonal antibodies, which bind S of SARS-CoV-2 with tight nanomolar to picomolar KD28,29,30,33; by comparison, WT sACE2 has only moderate affinity (KD ≈ 20 nM)16,43. To this end, mutagenic approaches, either targeted or through selection of diverse libraries of variants, provide a means for engineering decoy receptors with enhanced affinity.
In the first report44 that mutations within ACE2 can indeed be found that increase S affinity, Chan et al. performed deep mutagenesis on full length ACE243,45. A library of ACE2 variants was created that encoded all possible single amino acid variants at 117 sites, covering both the regions that interface with the RBD as well as the ACE2 active site. The library was then selected by fluorescence activated cell sorting (FACS) for ACE2 surface expression in human cells and tight binding to the RBD of SARS-CoV-2 protein S (Figure 2). ACE2 residues buried at the interface with S tend to be more conserved, while ACE2 residues at the interface periphery or within the active site were mutationally tolerant. Mutations of ACE2 residues N90 and T92, which form an N-glycosylation motif, are universally enriched for high binding affinity (with the exception of T92S), indicating that N90 glycosylation hinders binding, at least in this experiment’s cell line. Other mutations were also found dispersed across multiple sites that increase S binding, and some of these were rapidly screened as combinations of mutations in soluble ACE2 conjugated to superfolder GFP (sfGFP)46. Expression medium containing sACE2-sfGFP, without purification, can be incubated directly with cells expressing S and bound sACE2-sfGFP detected by flow cytometry. Ultimately, variants of sACE2 carrying four or three mutations (Figure 2C), dubbed sACE2.v2 and sACE2.v2.4, were engineered for an optimal balance of high affinity, expression and yield. Impressively, monomeric sACE2.v2, which spans ACE2 residues 19-615 that form the extracellular protease domain, outcompeted WT sACE2 fused to dimeric Fc of IgG1 and also competed effectively with anti-RBD Ig derived from Covid-19 positive patient sera. Soluble ACE2 forms a natural dimer (which we refer to as sACE22) if the construct is expanded up to ACE2 residues 730-740, which includes the dimerization/collectrin-like domain immediately C-terminal of the protease domain. Soluble ACE22 has increased apparent affinity and neutralization of authentic virus through avidity, demonstrating that each protease domain in sACE22 can bridge two S proteins. The leading variant from these efforts, dimeric sACE22.v2.4, had a picomolar KD for the SARS-CoV-2 RBD and achieved 50% relative infection inhibition of authentic virus at subnanomolar concentrations. Surprisingly, it showed similar neutralization efficacy against SARS-CoV-1 despite no consideration of binding to this virus during mutagenesis. This implied potential efficacy against other zoonotic coronaviruses that utilize ACE2 and may spill over to humans in the future. This was confirmed by demonstrating sACE22.v2.4 tightly binds diverse RBD sequences from SARS-related bat betacoronaviruses that use ACE2 as an entry receptor40. Furthermore, an in vitro selection of mutations within the RBD from SARS-CoV-2 failed to find S variants that lose affinity for the engineered sACE22.v2.4 decoy but retain binding to the wild type host ACE2 receptor, thus confirming the hypothesis that mechanisms for viruses to escape soluble decoy receptors are limited40.
In a similar deep mutagenesis experiment, Heinzelman and Romero investigated the influence on S protein binding of single-nucleotide variants (SNVs) in the ACE2 protease domain, evaluating nearly 4,000 amino acid substitutions47. Mutations were made using error-prone PCR, a rapid and reliable way of generating library diversity but which fails to capture multiple nucleotide changes within a single codon (unless error rates are excessively high). The library of the ACE2 protease domain was screened by yeast surface display (Figure 2B), and while most mutations decreased binding (it is, after all, easy to ’break’ a protein through random mutagenesis), approximately 4% of mutations were found to increase RBD binding. While mutations in the active site tended to have little effect (with the caveat that the background ACE2 sequence used for library generation already had catalytic activity knocked out), the authors found that residues in the chloride-binding site of ACE2, which regulates its peptidase activity48 and is over 40 Å from the RBD interface, do affect binding. In addition, a separate distal cluster of hydrophobic residues (L236, F588, L591, and L595) was also identified as a critical site affecting binding despite being over 30 Å from the RBD interface. By interrogating SNVs, the authors were also able to statistically evaluate the clinical and epidemiological relevance of ACE2 allelic variants in the human population, and they estimated that roughly 1 in 10,000 people may have SNVs that increase spike binding and roughly 4 in 1000 having SNVs that decrease spike binding. However, these predictions will need to be confirmed by genetics studies of patient cohorts, especially since mutations in ACE2 with altered affinity for S may also have other effects that impact infection in vivo, such as expression changes in human tissues. Overall, this work highlights the importance of considering long-range effects of mutations distal from the functional binding site.
The deep mutational scans of ACE2 are based on a single round of sequence diversification and selection, whereas others have employed multiple rounds to direct the in vitro evolution of ACE2 towards exceptionally tight affinity for SARS-CoV-2 S. Higuchi et al. focused mutagenesis to ACE2 residues 18-102 and 272-409 in the protease domain, respectively denoted as PD1 and PD2, that form the RBD interface49. Binding was assayed using the method previously developed for deep mutagenesis43, with human cells expressing the ACE2 library incubated with RBD-sfGFP and sorted by FACS. Mutations within the ”PD1” region were selected first, followed by mutagenesis within the ”PD2” region and additional rounds of selection, although additional mutations within the PD2 region did not substantially improve affinity. The ACE2 variant identified with highest affinity, termed 3N39 with 7 substitutions (Figure 2C), bound RBD with picomolar KD. Despite having a much higher mutational load than sACE2.v2.4, soluble 3N39-Fc fusions were soluble with minimal aggregation by size exclusion chromatography. The engineered 3N39-Fc fusion vastly outperformed the equivalent wild type ACE2-Fc fusion in virus neutralization.
The Kortemme, Hobman, and Wells groups went even further in using multiple rounds of engineering, diversification and selection to drive down the dissociation constant to the picomolar range50. Computational alanine scanning identified ACE2 residues H34, Q42 and K353 as hot spots that contribute disproportionately to the binding energy, and these residues with their neighbors were the focus for computational mutagenesis. Computationally designed ACE2 variants were purified as Fc fusions and binding to S was improved 3- to 11-fold. Four of these ACE2 variants served as parents that were then matured through yeast surface display, resulting in a further 14-fold improvement in apparent KD. Deep mutagenesis data from Chan et al43 was then considered, from which new ACE2 variants were designed with mutations that were not alanine scan-based hotspots and included sites outside the RBD interface that likely stabilize conformation. The final ACE2 variants had exceptionally tight affinity, with the lowest KD variants carrying 4-8 substitutions (Figure 2C). It was also independently confirmed that additional increases in apparent affinity can be achieved through inclusion of the ACE2 dimerization domain for avid binding and protein stabilization. It is worth noting that in this study deliberate action was taken to inactivate ACE2 enzymatic activity. This has historically been done by mutating two critical Zn2+-coordinating residues (H374N and H378N)51, but because these substitutions resulted in undesirable destabilization in the variant background, an alternative mutation (H345L within the substrate-binding cavity) was adopted. While the rationale for this is the elimination of vasodilatory effects from excess ACE2 peptidase activity that may cause hypotension, it is worth considering the potential benefits of keeping enzymatic activity (discussed below).
Pangolin (Pangolin-CoV-2020) and bat (isolate RaTG13) coronaviruses, in addition to human SARS-CoV-1 and -2, can initiate membrane fusion using not only human ACE2 but many other animal orthologs52. This informed the substitution of human ACE2-D30 for glutamate52, which is found in ACE2 of other species and is able to better reach K417 of the RBD for salt bridge formation16,17. In addition, inclusion of the dimerization domain together with the protease domain further facilitated the engineering of a sACE2-Ig fusion protein effective against multiple betacoronavirus strains. The discovery of the affinity-enhancing D30E mutation and benefits of including the dimerization domain overlap with independent work by others43,50. Another contributing factor was increased avidity (to be discussed further below) due to design of a tetrameric sACE2-Ig configuration52. Overall, it is apparent from all these studies that a truly effective therapeutic approach utilizing decoy ACE2 receptors will likely incorporate a multifaceted strategy of affinity enrichment, avidity and Fc fusions.
One final aspect to touch upon regarding affinity enhancement through mutagenesis is the connection between binding affinity and ACE2 enzymatic activity. For example, the sACE2.v2.4 variant has reduced catalytic activity even though its 3 amino acid substitutions are not within the active site43, thus demonstrating potentially unappreciated coupling between S affinity and ACE2 catalysis. Others have observed that some mutations that knock out ACE2 activity can increase S binding53, although many mutations in the active site have no effect on S affinity43,47. In particular, we highlight again the key finding by Heinzelman and Romero that mutations to a chloride-binding motif of ACE2, which is 40 Å from the RBD interface and is directly involved in regulating peptidase activity, can affect spike affinity, possibly due to conformational effects47. The binding of trimeric SARS-CoV-2 spike protein to wild type ACE2 also increases its peptidase activity 3- to 10-fold, with the SARS-CoV-2 RBD inducing bending of the N-terminal protease subdomain toward the C-terminal protease subdomain “…reminiscent of a closing dam”54. Although the precise mechanisms remain unclear, there are nonetheless multiple clues incriminating ACE2 conformation and dynamics on both catalytic activity and S affinity. We believe from a therapeutic perspective (to be discussed further below), maintaining catalytic activity is preferable due to the protective effects of ACE2 products on the pulmonary and cardiovascular systems.