ACE2 Fusions with Immunoglobulin Fc
Despite an ongoing clinical trial of wild type sACE2 in Europe42 and the studies reviewed above demonstrating the realization of picomolar affinity decoy receptors, it is likely that low sACE2 serum stability will limit its successful application as a therapeutic. The serum half-life of dimeric sACE2 in vivo is on the order of hours55,56, thereby requiring frequent dosing to maintain sACE2 in circulation. As a consequence, it is questionable whether sACE2 can be safely administered in an outpatient setting. Serum stability of biologic drugs is greatly increased by fusion of the therapeutic protein with the Fc region of IgG57. Serum proteins are turned over by constant internalization and lysosomal degradation, especially by endothelial cells. Internalized IgG-Fc engages the neonatal Fc receptor (FcRn) within endosomal compartments to direct its trafficking back to the plasma membrane, thereby returning the protein to the serum (Figure 3A). The previously highlighted works describing the discovery of high affinity sACE2 variants43,49,50,52 have already extended their findings to fusions with IgG1 Fc, and we believe that sACE2-Fc fusions will be the standard going forward for the production of high-affinity decoy receptors with excellent pharmacokinetics.
Recombinant human sACE2 has been developed over nearly two decades as a therapeutic for lung inflammation and acute respiratory distress syndrome (ARDS). Its administration has been shown to be protective against cardiovascular58 and pulmonary pathologies and to treat hypoxemia in multiple animal models59,60,61,62,63, and it has acceptable safety without causing episodes of hypotension in healthy and mechanically ventilated human subjects56,64. However, while sACE2 demonstrated the expected in vivo biochemical activity in ARDS patients, there was no statistically significant improvement in clinical outcomes in a phase 2 trial. The clinicians commented that “…lower plasma concentrations (of sACE2) over a longer duration may be more effective as a result of more sustained production of” ACE2 products64. These findings motivated the first published report of an sACE2-Fc fusion tested in vivo (in mice), in which the serum half-life was extended to over a week with substantially higher maximum serum activity63. Recombinant sACE2-Fc mitigated hypertension following injection of the vasopressor (and ACE2 substrate) angiotensin II for up to 7 days, whereas unfused sACE2 lost function in serum within hours. The Fc fusion protein reduced blood pressure and cardiac and renal fibrosis in a transgenic mouse model for hypertension and albuminuria in which renin is massively overexpressed63. This work demonstrated serum longevity of sACE2 fused to IgG1-Fc as well as the potent physiological effects of ACE2 catalytic activity.
More recently with the emergence of SARS-CoV-2, multiple groups have explored IgG1-Fc fusions of sACE2 and consistently find long serum half-life without adverse toxicity in small animals53,38. The Fc region of IgG subclasses engage multiple Fc receptors to effect function, including not only FcRn to mediate serum stability and uptake into the lungs, but also a suite of inflammatory Fc γ receptors (FcγR)65. These receptors are expressed on a variety of cell types, especially in the immune system, and are responsible for effector functions such as antibody-dependent cellular cytotoxicity, cytokine release, antigen presentation and co-stimulation (Figure 3A). Until different Fc fusion proteins are properly assessed in controlled in vivo experiments, it is unclear whether these effector functions will aid in clearance and recovery from SARS-CoV-2 infection. Iwanaga et al have hypothesized that interactions of a sACE2-Fc biologic with inflammatory FcγRs may exacerbate inflammation and cytokine storms in COVID-19 patients53. Consequently, two leucine residues in the IgG1-Fc moiety were mutated to alanines (L234A and L235A). These so-called LALA mutations disrupt FcγR interactions while leaving FcRn binding intact for high serum stability. The LALA variant of sACE2-Fc had long serum half-life, was distributed to lung tissues, and retained virus neutralization efficacy.