ACE2 COMPOSITIONS AND METHODS
This disclosure describes recombinant angiotensin-converting enzyme II (ACE2) polypeptides, fusion proteins, and compositions thereof having improved binding affinity for the SARS-CoV-2 spike protein receptor binding domain relative to wild-type ACE2. Also provided are methods of using the recombinant ACE2 polypeptides, fusion proteins, and compositions thereof for treating subjects infected with a SARS-CoV-2 virus (i.e., subjects with COVID-19), subjects having symptoms suggestive of a SARS-CoV-2 infection, and subjects exposed to or at risk of exposure to SARS-CoV-2 virus. Other virus infections may also be treated.
1 . A recombinant ACE2 polypeptide comprising a soluble ACE2 receptor ectodomain polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2 or 3 and comprising at least one amino acid residue substitutions selected from the group consisting of Q18R, S19P, A25V, T27A, T27Y, K31F, K31Y, N33D, N33S, H34A, H34I, H34S, H34V, E35Q, F40D, F40L, F40S, Q42L, N49D, N49S, N51S, N53S, E57G, N61D, M62T, M62I, M62V, N64D, K68R, W69R, W69V, W69K, W69I, Q76R, L79P, L79F, L79T, N90Q, L91P, L100P, and Q101R, wherein the residues are numbered with reference to SEQ ID NO:1.
2 . The recombinant ACE2 polypeptide of claim 1 , wherein the soluble ACE2 receptor ectodomain polypeptide comprises at least two amino acid residue substitutions selected from the group consisting of Q18R, S19P, A25V, T27A, T27Y, K31F, K31Y, N33D, N33S, H34A, H34I, H34S, H34V, E35Q, F40D, F40L, F40S, Q42L, N49D, N49S, N51S, N53S, E57G, N61D, M62T, M62I, M62V, N64D, K68R, W69R, W69V, W69K, W69I, Q76R, L79P, L79F, L79T, N90Q, L91P, L100P, and Q101R, wherein the residues are numbered with reference to SEQ ID NO:1.
3 . The recombinant ACE2 polypeptide of claim 1 , wherein the soluble ACE2 receptor ectodomain polypeptide comprises amino acid residue substitutions:
i. K31F, N33D, H34S, and E35Q;
ii. K31F, N33D, H34A, E35Q, N49D, N51S, N53S, E57G, and N64D;
iii. T27A, K31F, N33D, H34S, E35Q, N61D, K68R, and L79P;
iv. S19P, N33S, H34V, F40L, N49D, and L100P;
v. K31F, N33D, H34S, E35Q, W69R, and Q76R;
vi. Q18R, K31F, N33D, H34S, E35Q, W69R, and Q76R;
vii. Q18R, K31F, N33D, H34S, E35Q, W69V, and Q76R;
viii. Q18R, K31F, N33D, H34S, E35Q, W69K, and Q76R;
ix. Q18R, K31F, N33D, H34S, E35Q, W69I, and Q76R;
x. T27A, H34A, N49S, V59A, N63S, K68R, E75G, N90Q, and Q103R;
xi. K31F, N33D, H34T, N53D, W69R, and E75K;
xii. S19P, K26R, T27A, H34A, S44G, and M62T;
xiii. K31F, H34I, E35Q, and N90Q;
xiv. A25V, T27A, H34A, and F40D;
xv. K31Y, W69V, L79T, and L91P;
xvi. T27Y, H34A, and N90Q;
xvii. S19P, Q42L, L79T, and N90Q;
xviii. K31F, H34I, E35Q; or
xix. H34V and N90Q,
wherein the residues are numbered with reference to SEQ ID NO:1.
4 . The recombinant ACE2 polypeptide of claim 1 , wherein the soluble ACE2 receptor ectodomain polypeptide comprises amino acid residue substitutions H374N and H378N.
5 . The recombinant ACE2 polypeptide of claim 1 , wherein the soluble ACE2 receptor ectodomain polypeptide comprises amino acid residue substitution H345L.
6 . A fusion protein comprising the recombinant ACE2 polypeptide of claim 1 fused to a dimerization domain.
7 . The fusion protein of claim 6 , wherein the recombinant ACE2 polypeptide is fused to the dimerization domain via a peptide linker.
8 . The fusion protein of claim 6 , wherein the dimerization domain comprises an Fc domain.
9 . A recombinant nucleic acid encoding the recombinant ACE2 polypeptide protein of claim 1 or the fusion protein of claim 6 .
10 . A DNA construct comprising a promoter operably linked to the recombinant nucleic acid of claim 9 .
11 . The DNA construct of claim 10 , wherein the promoter is a heterologous promoter.
12 . A vector comprising the DNA construct of claim 10 .
13 . A host cell comprising the recombinant nucleic acid of claim 9 .
14 . A host cell comprising the DNA construct of claim 10 .
15 . A host cell comprising the vector of claim 12 .
16 . The host cell of claim 13 , wherein the host cell is a eukaryotic cell.
17 . A composition comprising a dimer of the recombinant ACE2 polypeptide of claim 1 .
18 . A method of producing a recombinant ACE2 polypeptide comprising culturing the host cell of claim 13 under conditions sufficient for the production of the recombinant ACE2 polypeptide by the host cell.
19 . A pharmaceutical preparation comprising:
(a) the recombinant ACE2 polypeptide of claim 1 or the fusion protein of claim 6 ; and
(b) a pharmaceutically acceptable carrier.
20 . A method for treating a subject infected with a SARS-CoV-2 virus or having symptoms suggestive of a SARS-CoV-2 infection, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical preparation of claim 19 .
21 . The method of claim 20 , wherein the subject has a confirmed SARS-CoV-2 infection.
22 . A method for treating a subject exposed to a SARS-CoV-2 virus or at risk of exposure to SARS-CoV-2 virus, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical preparation of claim 19 .
23 . The method of claim 20 , wherein the subject is human.
24 . The method of claim 20 , wherein the pharmaceutical preparation is administered intravenously.
25 . The method of claim 20 , wherein the pharmaceutical preparation is administered at least once per day.
26 . (canceled)
27 . The recombinant ACE2 polypeptide, fusion protein, and/or composition of claim 26 , wherein the recombinant ACE2 polypeptide, the fusion protein, and/or the composition has greater than 180-fold higher affinity for SARS-CoV-2 spike RBD as compared to wild-type human ACE2 protein ectodomain.
28 - 29 . (canceled)
30 . A composition comprising the fusion protein of claim 1 .
31 . The recombinant ACE2 polypeptide of claim 1 , wherein the recombinant ACE2 polypeptide, has increased binding affinity for SARS-CoV-2 spike RBD and/or has increased efficacy in neutralizing SARS-CoV-2 virus relative to wild-type human ACE2 protein ectodomain.
32 . The fusion protein of claim 6 , wherein the fusion protein has increased binding affinity for SARS-CoV-2 spike RBD and/or has increased efficacy in neutralizing SARS-CoV-2 virus relative to wild-type human ACE2 protein ectodomain.
33 . The composition of claim 17 , wherein the fusion protein has increased binding affinity for SARS-CoV-2 spike RBD and/or has increased efficacy in neutralizing SARS-CoV-2 virus relative to wild-type human ACE2 protein ectodomain.
34 . The recombinant ACE2 polypeptide of claim 31 , wherein the recombinant ACE2 polypeptide has greater than 25-fold higher neutralization efficacy for SARS-CoV-2 virus compared to wild-type human ACE2 protein ectodomain.
35 . The fusion protein of claim 32 , wherein the fusion has greater than 25-fold higher neutralization efficacy for SARS-CoV-2 virus compared to wild-type human ACE2 protein ectodomain.
36 . The composition of claim 33 , wherein the fusion has greater than 25-fold higher neutralization efficacy for SARS-CoV-2 virus compared to wild-type human ACE2 protein ectodomain.