ACE2 muteins and methods of using the same
The invention relates generally to non-naturally occurring ACE2 proteins and their use in the treatment of coronavirus infection.
1 . A fusion protein comprising a human angiotensin-converting enzyme 2 (ACE2) mutein and an antibody Fc domain, wherein the human ACE2 mutein comprises an amino acid sequence at least 80% identical to amino acids 19-615 of wild-type human ACE2 (SEQ ID NO: 1), wherein the fusion protein has a melting temperature of at least 60° C. in a differential scanning fluorimetry (DSF) assay, and wherein the ACE2 mutein is not identical to a wild-type ACE2 of a non-human animal.
2 . The fusion protein of claim 1 , comprising:
a. two or more substitutions of a non-cysteine amino acid in wild-type human ACE2 (SEQ ID NO: 1) to a cysteine amino acid;
b. two or more amino acid substitutions, which are disposed on opposite sides of the angiotensin II substrate-binding cleft of the ACE2 mutein; and/or
c. one or more amino acid substitutions, which increase the volume of buried hydrophobic amino acids and/or decrease the volume of exposed or partially-exposed hydrophobic amino acids in comparison to wild-type human ACE2 (SEQ ID NO:1).
3 . The fusion protein of claim 2 , wherein one of said cysteine amino acids is disposed on one side of the angiotensin II substrate-binding cleft of the ACE2 mutein and the other one of said cysteine amino acids is disposed on the opposite side of the cleft, wherein said cysteine amino acid substitutions form a disulfide bond.
4 . The fusion protein of claim 1 , comprising the substitutions S43C/G66C, K74C/S106C, S128C/V343C, and/or A714C of wild-type human ACE2 (SEQ ID NO: 1).
5 . The fusion protein of claim 1 , comprising at least one substitution at position H345 or F504.
6 . The fusion protein of claim 1 , wherein said ACE2 mutein comprises the substitution H345Y.
7 . The fusion protein of claim 1 , comprising one or more substitutions at a position corresponding to I259, C261, V339, A342, and/or C498 of SEQ ID NO: 1.
8 . The fusion protein of claim 7 , comprising one or more of the substitutions I259T, C261P, V339G, A342V, and/or C498M.
9 . The fusion protein of claim 1 , wherein the ACE2 mutein comprises a collectrin domain having an amino acid sequence at least 80% identical to the collectrin domain of wild-type human ACE2 (SEQ ID NO: 1) amino acids 616-723.
10 . The fusion protein of claim 1 , comprising one or more substitutions at a position corresponding to V620, I622, A650, L656, M662, L664, V685, and I695 of SEQ ID NO: 1.
11 . The fusion protein of claim 10 , comprising one or more of the substitutions V620I, I622L, A650V, A650I, L656S, M662G, M662D, L664G, L664P, V685L, and/or I695T.
12 . The fusion protein of claim 1 , comprising a substitution at one or more of the positions corresponding to N90, L91, and T92.
13 . The fusion protein of claim 12 , comprising the substitution N90D.
14 . The fusion protein of claim 1 , comprising the substitution A25V.
15 . A nucleic acid encoding the fusion protein of claim 1 .
16 . A cell comprising the nucleic acid of claim 15 .
17 . A composition comprising the fusion protein of claim 1 .
18 . The fusion protein of claim 1 , wherein the melting temperature is between 60° C. and 70° C.
19 . The fusion protein of claim 1 , wherein the melting temperature is between 60° C. and 65° C.