Recombinant metapneumovirus f proteins and their use

Patent metadata

Field	Value
Publication number	US-2024024458-A1
Application number	US-202318462340-A
Country	US
Kind code	A1
Filing date	Sep 6, 2023
Priority date	Dec 24, 2014
Publication date	Jan 25, 2024
Grant date	—

Abstract

Metapneumovirus (MPV) F proteins stabilized in a prefusion conformation, nucleic acid molecules and vectors encoding these proteins, and methods of their use and production are disclosed. In several embodiments, the MPV F proteins and/or nucleic acid molecules can be used to generate an immune response to MPV in a subject. In additional embodiments, the therapeutically effective amount of the MPV F ectodomain trimers and/or nucleic acid molecules can be administered to a subject in a method of treating or preventing MPV infection.

First claim

We claim:

1 . A method for generating an immune response to metapneumovirus (MPV) F protein in a subject, comprising:
administering to the subject an effective amount of a nucleic acid molecule encoding a recombinant MPV F protein or immunogenic fragment thereof stabilized in a prefusion conformation by one or more amino acid substitutions compared to a native MPV F protein sequence, wherein the recombinant MPV F protein comprises a F 2 polypeptide and a F 1 ectodomain, wherein the one or more amino acid substitutions introduce one or more non-native intra- or inter-protomer disulfide bonds that, alone or in combination with other modifications, stabilize the MPV F protein in the prefusion conformation.

2 . The method of claim 1 , wherein the recombinant MPV F protein comprises a non-natural disulfide bond between A113C and A339C cysteine substitutions that stabilizes the recombinant MPV F protein in the prefusion conformation, wherein the amino acid positions correspond to a reference MPV F sequence set forth as SEQ ID NO: 7.

3 . The method of claim 1 , wherein the recombinant MPV F protein comprises:
a non-natural disulfide bond between A120C and Q426C cysteine substitutions that stabilizes the recombinant MPV F protein in the prefusion conformation; or a non-natural disulfide bond between A120C and Q428C cysteine substitutions that stabilizes the recombinant MPV F protein in the prefusion conformation; and wherein the amino acid positions correspond to a reference MPV F sequence set forth as SEQ ID NO: 7.

4 . The method of claim 1 , wherein the recombinant MPV F protein comprises one or more cavity filling amino acid substitutions that reduce the volume of a threonine 160 cavity and/or an isoleucine 177 cavity, wherein the amino acid positions correspond to a reference MPV F sequence set forth as SEQ ID NO: 7.

5 . The method of claim 4 , wherein the recombinant MPV F protein comprises:
one or more cavity filling amino acid substitutions at positions 160, 162, and/or 157 to reduce the volume of the threonine 160 cavity; and/or one or more cavity filling amino acid substitutions at positions 177, 58, 169, 54, and/or 55 to reduce the volume of the isoleucine 177 cavity; and wherein the amino acid positions correspond to a reference MPV F sequence set forth as SEQ ID NO: 7.

6 . The method of claim 4 , wherein the cavity filling amino acid substitution comprises a F, L, W, Y, H, or M substitution.

7 . The method of claim 4 , wherein the recombinant MPV F protein comprises a T160F substitution, a I177L substitution, or T160F and I177L substitutions that stabilize the recombinant MPV F protein in the prefusion conformation, wherein the amino acid positions correspond to a reference MPV F sequence set forth as SEQ ID NO: 7.

8 . The method of claim 1 , wherein the recombinant MPV F protein comprises a non-natural disulfide bond between A113C and A339C cysteine substitutions, and T160F and I177L cavity filling substitutions, that stabilize the recombinant MPV F protein in the prefusion conformation, wherein the amino acid positions correspond to a reference MPV F sequence set forth as SEQ ID NO: 7.

9 . The method of claim 8 , further comprising one or more additional amino acid substitutions.

10 . The method of claim 1 , wherein the recombinant MPV F protein comprises a proline amino acid substitution at one of positions 183-189 that reduces or prevents formation of an oc7 helix to stabilize the MPV F protein in the prefusion conformation, wherein the amino acid positions correspond to a reference MPV F sequence set forth as SEQ ID NO: 7.

11 . The method of claim 10 , comprising the A185P substitution.

12 . The method of claim 1 , wherein the recombinant MPV F protein is not glycosylated at N57, N172, or N57 and N172, wherein the amino acid positions correspond to a reference MPV F sequence set forth as SEQ ID NO: 7.

13 . The method of claim 12 , wherein the recombinant MPV F protein comprises one of:
a N57Q substitution; a N172Q substitution; a N57Q substitution and a N172Q substitution, a T59A substitution; a T174A substitution; or a T59A substitution and a T174A substitution; to remove N57 and/or N172 N-glycan sequons; and
wherein the amino acid positions correspond to a reference MPV F sequence set forth as SEQ ID NO: 7.

14 . The method of claim 1 , wherein the recombinant MPV F protein comprises:
one or more cysteine substitutions that introduce the non-native intra-protomer disulfide bond between the amino acids of:
any one of positions 103-120 and any one of positions 335-345;
any one of positions 107-118 and any one of positions 335-342;
any one of positions of α-helix 3 (residues 117-129) and any one of positions of β-strand 6 (residues 256-261);
any one of positions of α-helix 2 and any one of positions adjacent to the cleavage site (residues 87-102) with α-helix 3 (residues 117-127);
any one of positions 102-113 and any one of positions of α-helix 3 (residues 117-127); or
any one of positions 102-113 and any one of positions of α-helix 2 and residues adjacent to the cleavage site (residues 87-102); and/or
one or more cysteine substitutions that introduce the non-native inter-protomer disulfide bond between the amino acids of:
any one of positions 337-341 and any one of positions 421-426;
any one of positions 112-120 and any one of positions 424-432;
any one of positions 150-156 with the addition of a glycine residue, and any one of positions 392-400;
any one of positions 112-120 and any one of positions 370-377;
any one of positions 365-375 and any one of positions 455-465;
any one of positions 365-375 and any one of positions 105-115; or
any one of positions 60-70 and any one of positions 175-185;
wherein the positions correspond to the amino acids positions of a reference MPV F protein sequence set forth as SEQ ID NO: 7.

15 . The method of claim 1 , wherein the recombinant MPV F protein comprises one of the following combinations of substitutions:
A113C, A339C, and D183P; A113C, A339C, and A185P; A113C, A339C, and D186P; A113C, A339C, T160F, I177L, and D183P; A113C, A339C, T160F, I177L, and A185P; A113C, A339C, T160F, I177L, and D186P; A113C, A339C, A120C, Q426C, T160F, I177L, and D183P; A113C, A339C, A120C, Q428C, T160F, I177L, and D183P; A113C, A339C, A120C, Q426C, T160F, I177L, and A185P; A113C, A339C, A120C, Q428C, T160F, I177L, and A185P; A113C, A339C, A120C, Q426C, T160F, I177L, and D186P; A113C, A339C, A120C, Q428C, T160F, I177L, and D186P; A113C, A339C, D183P, and N57Q; A113C, A339C, A185P, and N57Q; A113C, A339C, D186P, and N57Q; A113C, A339C, T160F, I177L, D183P, and N57Q; A113C, A339C, T160F, I177L, A185P, and N57Q; A113C, A339C, T160F, I177L, D186P, and N57Q; A113C, A339C, A120C, Q426C, T160F, I177L, D183P, and N57Q; A113C, A339C, A120C, Q428C, T160F, I177L, D183P, and N57Q; A113C, A339C, A120C, Q426C, T160F, I177L, A185P, and N57Q; A113C, A339C, A120C, Q428C, T160F, I177L, A185P, and N57Q; A113C, A339C, A120C, Q426C, T160F, I177L, D186P, and N57Q; A113C, A339C, A120C, Q428C, T160F, I177L, D186P, and N57Q; A113C, A339C, D183P, and N172Q; A113C, A339C, A185P, and N172Q; A113C, A339C, D186P, and N172Q; A113C, A339C, T160F, I177L, D183P, and N172Q; A113C, A339C, T160F, I177L, A185P, and N172Q; A113C, A339C, T160F, I177L, D186P, and N172Q; A113C, A339C, A120C, Q426C, T160F, I177L, D183P, and N172Q; A113C, A339C, A120C, Q428C, T160F, I177L, D183P, and N172Q; A113C, A339C, A120C, Q426C, T160F, I177L, A185P, and N172Q; A113C, A339C, A120C, Q428C, T160F, I177L, A185P, a

Assignees

• The Us Secretary Department Of Health And Human Servic
• Inst Res Biomedicine

Inventors

• Kwong Peter
• Joyce Michael Gordon
• Zhang Baoshan
• Yang Yongping
• Collins Peter
• Buchholz Ursula
• Corti Davide
• Lanzavecchia Antonio
• Stewart-Jones Guillaume

Classifications

• A61K39/155 Primary

  Paramyxoviridae, e.g. parainfluenza virus · CPC title

• A61K39/39

  characterised by the immunostimulating additives, e.g. chemical adjuvants · CPC title

• C07K14/005

  from viruses · CPC title

• A61K48/00

  Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy · CPC title

• C07K14/135

  Respiratory syncytial virus · CPC title