Introduction
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a significant threat to public health and a great burden on the global economy1. SARS-CoV-2 and severe acute respiratory syndrome coronavirus (SARS-CoV) have 79.5% homologous sequences, and their S protein has 76.5% similarity in amino acid sequences2. Based on previous studies, researchers quickly identified angiotensin-converting enzyme 2 (ACE2) as a key receptor for SARS-CoV-21,3–5. ACE2 plays a key negative regulatory role in the renin-angiotensin system (RAS)6,7 and promotes amino acid transport in the kidney and intestine under normal physiological conditions5. ACE2 is essential for SARS-CoV-2 to invade human cells, and during infection, the receptor-binding domain (RBD) on the S1 subunit of SARS-CoV-2 binds to ACE2, which then fuses the cell membrane of the virus to the host cell, thereby mediating viral entry into the host cell8. However, ACE2 deficiency may aggravate the prognosis of patients with COVID-199, especially those with underlying diseases (such as hypertension, heart disease, cirrhosis and cancer)10,11.
Blocking spike/ACE2 conjugation is a central strategy for vaccine design and multiple therapeutics12. With the development of drugs for managing COVID-19, patient outcomes have improved significantly. However, in some elderly people with underlying diseases, SARS-CoV-2 continues to pose a severe threat; additionally, herd immunity is low, it is difficult to produce an effective response to the vaccine, and the neutralization effect of monoclonal antibodies is hindered because SARS-CoV-2 hides its RBD after infection in the body13–16. Moreover, as the virus evolves, variants continue to emerge, which not only affects the infectivity of SARS-CoV-2 but also reduces the efficacy of vaccines, convalescent serum, and monoclonal antibody therapy17–21. Therefore, ACE2 is an excellent drug target because regardless of the number of SARS-CoV-2 variants, the virus relies on ACE2 to infect humans2,22.
Recombinant human ACE2 (rhACE2) can be used as a supplement to ACE2 in the body, which can competitively block SARS-CoV-2 infection23,24. Preclinical research has revealed that rhACE2 protects mice from severe acute lung injury by inhibiting the binding of SARS coronavirus to its own ACE2 in lung cells1,25 and that rhACE2 can inhibit SARS-CoV-2 infection in artificial human tissues26. Clinical trials (NCT04335136) have demonstrated that soluble rhACE2 (APN01) binds to the RBD and full-length spike protein of the SARS-CoV-2 variant and is effective in neutralizing infection in all test variants27. Furthermore, another study obtained good results in animal experiments by in situ administration of APN01 in the respiratory tract in the form of aerosol inhalation, and a phase I clinical trial (NCT05065645) is currently ongoing28. Nevertheless, a clinical trial to assess the efficacy of rhACE2 in COVID-19 patients in China (NCT04287686) was withdrawn29 due to concerns that ACE2 infusion may reduce circulating Ang II and increase Ang-(1-7) levels, leading to possible complications such as hypotension in patients with COVID-19 in the late stages of the disease30. Our current study may alleviate this concern, as we can accurately reflect in situACE2 levels in organs through PET imaging, which can help in evaluating the efficacy of rhACE2 and provide a scientific basis for rhACE2 therapy.
Existing methods such as immunohistochemistry and serological testing cannot be used to detect the dynamic expression level of ACE2 and its spatial distribution in vivo , which is an obstacle to further development of ACE2 as a biomarker for the diagnosis and treatment of COVID-19. Molecular imaging, as a noninvasive method, can be used to quantitatively monitor the spatial distribution of molecular targetsin vivo with high sensitivity. Positron emission tomography (PET) has been widely used to detect disease-related biomarkers using radioligands.
DX600 is an ACE2-specific inhibitory peptide found in the peptide library based on restriction phages with a Ki as low as 2.8 nM and is highly specific for ACE231. For radionuclide68Ga labeling, we coupled DX600 with the bifunctional chelate DOTA, named HZ20. In our previous study32, HZ20 was shown to target rhACE2 with high affinity and specificity, and the Kd values of the SPR assay for DX600 and HZ20 were 98.7 and 100.0 nM, respectively. Additionally, we used 68Ga-HZ20 to assess the background level of ACE2 and the in vivo distribution and expression levels in 20 healthy volunteers of different ages and sexes and one COVID-19-rehabilitated person32. We also developed a neutralized nanobody-based radiotracer,68Ga-Nb1159. It can be used to visualize the localization and distribution of the SARS-CoV-2 RBD with a high target-to-background ratio33. Furthermore, there have been no reports of radiotracer detection of the distribution and content of rhACE2 in different organs.
Inspired by the previous ACE2/RBD targeting probes and the recent eased restrictions, we aimed to noninvasively map rhACE2 in the body in various tissue microenvironments. We optimized the labeling conditions of radionuclide 68Ga and obtained the radioactive probe 68Ga-HZ20 with a higher labeling rate and yield. We also constructed rhACE2 in situ models in different organs of mice, for example, liver, spleen, brain and tumor tissues, and used68Ga-HZ20 to monitor the content and distribution of exogenous rhACE2 in situ in model mouse organs for rhACE2 therapy.