Catalysis deactivated angiotensin-converting enzyme 2 (ACE2) variants and their uses
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Publication Number
US-11453869-B2
Publication Date
2022-09-27
Expiration Date
Abstract
Angiotensin-converting enzyme 2 (ACE2) has been confirmed as a specific receptor for several (3 group coronaviruses include severe respiratory syndrome (SARS) coronavirus (SARS-CoV-1) and recently the causative agent for the World pandemic CoVID-19, SARS-CoV-2, and low pathogenic coronavirus of HCoV-NL63, a member in α-coronavirus group. Viral spike protein (S) of viral envelope is confirmed to bind to ACE2 as viral receptor to start a virus replication cycle. The present invention provides ACE2 and its mutants or variants, the viral or non-viral vectors thereof. Methods of treatment of viral infection of a human subject by using such mutants or variants are also provided.
Core Innovation
The invention provides an isolated angiotensin converting enzyme 2 (ACE2) polypeptide containing one or more mutations relative to wild-type ACE2 that cause loss of ACE2 enzymatic activity compared to the wild-type ACE2. The polypeptide comprises an amino acid sequence selected from SEQ ID NOs 3, 4, 10, 17, and 18.
The disclosed ACE2 polypeptides are catalytically deactivated ACE2 extracellular domain (ACE2-vECD) mutants designed to abolish ACE2 enzymatic activity by disrupting the zinc-binding catalytic motif, while retaining or enhancing binding to coronavirus spike protein S1. The catalytic-site disruption region is associated with HEXXHE/HEXXH…E and specific catalytic-site residues including H374, E375, H378, and E402.
The mutants are presented as engineered ACE2-vECD-Fc fusion constructs, including ACE2-vECD fused to an IgG Fc. The mutant Fc fusions lose enzymatic RFU activity (>99.9% depletion) while maintaining binding to SARS-CoV-1, SARS-CoV-2, and MERS-CoV S1, including increased binding affinity for SARS-CoV-2 S1 for certain variants and enhanced binding to MERS-CoV.
Claims Coverage
The independent claim covers isolated mutated ACE2 polypeptides with one or more mutations relative to wild-type ACE2 that cause loss of ACE2 enzymatic activity and whose amino acid sequences are selected from SEQ ID NOs 3, 4, 10, 17, and 18. Dependent claims narrow the construct by incorporating the mutated ACE2 into N- or C-terminal fusion proteins with immune-cell-targeting peptides that bind FcγR on lymphocytes, with further restriction to T cells, B cells, or natural killer cells and specification of the targeting peptide as an Fc domain of human IgG antibodies.
Mutated ACE2 polypeptide causing loss of ACE2 enzymatic activity
An isolated angiotensin converting enzyme 2 (ACE2) polypeptide comprising one or more mutations relative to the wild-type ACE2 that cause the loss of ACE2 enzymatic activity comparing to the wild-type ACE2.
Mutated ACE2 polypeptide amino acid sequence selected from SEQ ID NOs 3, 4, 10, 17, and 18
The polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 3, 4, 10, 17, and 18.
N- or C-terminal fusion with immune-cell-targeting peptide
A fusion protein including the isolated mutated ACE2 polypeptide fused at the N- or C-terminus to a peptide capable of binding a receptor on an immune system-associated cell.
FcγR-binding peptide for immune-cell lymphocytes
The fusion protein includes a peptide that is a ligand binding an FcγR on an immune cell of lymphocyte.
Lymphocyte type selected as T, B, or natural killer cells
The immune lymphocyte cell is selected from T cells, B cells, or natural killer cells.
Human IgG Fc domain as the targeting peptide
The peptide is a Fc domain of human IgG antibodies (Fcγ).
Overall, the claim coverage centers on isolated mutated ACE2 polypeptides with loss of ACE2 enzymatic activity and specified sequence sets (SEQ ID NOs 3, 4, 10, 17, and 18), optionally formatted as N- or C-terminal fusion proteins with peptides that bind FcγR on specific lymphocyte types (T, B, or natural killer cells), including embodiments where the targeting peptide is a human IgG Fc domain.
Stated Advantages
The engineered ACE2-vECD Fc fusions abolish ACE2 enzymatic activity, with enzymatic RFU activity depleted by more than 99.9%.
The catalytically deactivated ACE2 mutants maintain binding to coronavirus spike protein S1, including SARS-CoV-1, SARS-CoV-2, and MERS-CoV S1.
The mutants can exhibit increased binding affinity to SARS-CoV-2 S1 for certain variants.
The partial content reports virus neutralization activity with IC50 values in a stated range (~5–35.5 µg/mL).
Documented Applications
Therapeutic and prophylactic use as virus-receptor blockers based on ACE2 binding to coronavirus spike protein S1.
In vitro neutralization assays using SARS-CoV-2 pseudovirus and virulent virus neutralization metrics (IC50 values are reported in the partial content).
Delivery using viral vectors, including AAV (AAV5 and AAV-ACE2-vECD-Fc are mentioned in the partial content).
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