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.