# US 11,027,007 B2 · Recombinant metapneumovirus F proteins and their use

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- Number: US 11,027,007 B2
- Document ID: US11027007B2
- Status: Granted
- Kind: B2
- Application number: 16/578,748
- Filed: 2019-09-23
- Published: 2020-01-09
- Granted: 2021-06-08
- Earliest claimed: 2014-12-24
- Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services; Institute for Research in Biomedicine
- Applicants: The United States of America, as represented by the Secretary, Department of Health and Human Services; Institute for Research in Biomedicine
- Inventors: Peter Kwong; Michael Gordon Joyce; Baoshan Zhang; Yongping Yang; Peter Collins; Ursula Buchholz; Davide Corti; Antonio Lanzavecchia; Guillaume Stewart-Jones
- Web page: https://patentgrep.com/patent/US11027007B2

## CPC

- A61K 39/155: Paramyxoviridae, e.g. parainfluenza virus
- A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C07K 14/005: from viruses
- C07K 14/135: Respiratory syncytial virus
- C07K 2319/70: containing domain for protein-protein interaction
- C07K 2319/73: containing coiled-coiled motif (leucine zippers)
- C12N 2760/18322: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2760/18334: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/18522: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2760/18534: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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.

## Claims

### Claim 1

A nucleic acid molecule encoding a recombinant metapneumovirus (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 F2 polypeptide and a F1 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.

### Claim 2 (depends on 1)

The nucleic acid molecule 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.

### Claim 3 (depends on 1)

The nucleic acid molecule 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.

### Claim 4 (depends on 1)

The nucleic acid molecule 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.

### Claim 5 (depends on 4)

The nucleic acid molecule 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.

### Claim 6 (depends on 4)

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

### Claim 7 (depends on 4)

The nucleic acid molecule 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.

### Claim 8 (depends on 1)

The nucleic acid molecule 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.

### Claim 9 (depends on 8)

The nucleic acid molecule of claim 8, further comprising one or more additional amino acid substitutions.

### Claim 10 (depends on 1)

The nucleic acid molecule 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 α7 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.

### Claim 11 (depends on 10)

The nucleic acid molecule of claim 10, comprising the A185P substitution.

### Claim 12 (depends on 1)

The nucleic acid molecule 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.

### Claim 13 (depends on 12)

The nucleic acid molecule 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.

### Claim 14 (depends on 1)

The nucleic acid molecule 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.

### Claim 15 (depends on 1)

The nucleic acid molecule 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, and N172Q;
A113C, A339C, A120C, Q426C, T160F, I177L, D186P, and N172Q;
A113C, A339C, A120C, Q428C, T160F, I177L, D186P, and N172Q;
A113C, A339C, D183P, N57Q, and N172Q;
A113C, A339C, A185P, N57Q, and N172Q;
A113C, A339C, D186P, N57Q, and N172Q;
A113C, A339C, T160F, I177L, D183P, N57Q, and N172Q;
A113C, A339C, T160F, I177L, A185P, N57Q, and N172Q;
A113C, A339C, T160F, I177L, D186P, N57Q, and N172Q;
A113C, A339C, A120C, Q426C, T160F, I177L, D183P, N57Q, and N172Q;
A113C, A339C, A120C, Q428C, T160F, I177L, D183P, N57Q, and N172Q;
A113C, A339C, A120C, Q426C, T160F, I177L, A185P, N57Q, and N172Q;
A113C, A339C, A120C, Q428C, T160F, I177L, A185P, N57Q, and N172Q;
A113C, A339C, A120C, Q426C, T160F, I177L, D186P, N57Q, and N172Q;
A113C, A339C, A120C, Q428C, T160F, I177L, D186P, N57Q, and N172Q;
A113C, A339C, D183P, and T59A;
A113C, A339C, A185P, and T59A;
A113C, A339C, D186P, and T59A;
A113C, A339C, T160F, I177L, D183P, and T59A;
A113C, A339C, T160F, I177L, A185P, and T59A;
A113C, A339C, T160F, I177L, D186P, and T59A;
A113C, A339C, A120C, Q426C, T160F, I177L, D183P, and T59A;
A113C, A339C, A120C, Q428C, T160F, I177L, D183P, and T59A;
A113C, A339C, A120C, Q426C, T160F, I177L, A185P, and T59A;
A113C, A339C, A120C, Q428C, T160F, I177L, A185P, and T59A;
A113C, A339C, A120C, Q426C, T160F, I177L, D186P, and T59A;
A113C, A339C, A120C, Q428C, T160F, I177L, D186P, and T59A;
A113C, A339C, D183P, and T174A;
A113C, A339C, A185P, and T174A;
A113C, A339C, D186P, and T174A;
A113C, A339C, T160F, I177L, D183P, and T174A;
A113C, A339C, T160F, I177L, A185P, and T174A;
A113C, A339C, T160F, I177L, D186P, and T174A;
A113C, A339C, A120C, Q426C, T160F, I177L, D183P, and T174A;
A113C, A339C, A120C, Q428C, T160F, I177L, D183P, and T174A;
A113C, A339C, A120C, Q426C, T160F, I177L, A185P, and T174A;
A113C, A339C, A120C, Q428C, T160F, I177L, A185P, and T174A;
A113C, A339C, A120C, Q426C, T160F, I177L, D186P, and T174A;
A113C, A339C, A120C, Q428C, T160F, I177L, D186P, and T174A;
A113C, A339C, D183P, T59A, and T174A;
A113C, A339C, A185P, T59A, and T174A;
A113C, A339C, D186P, T59A, and T174A;
A113C, A339C, T160F, I177L, D183P, T59A, and T174A;
A113C, A339C, T160F, I177L, A185P, T59A, and T174A;
A113C, A339C, T160F, I177L, D186P, T59A, and T174A;
A113C, A339C, A120C, Q426C, T160F, I177L, D183P, T59A, and T174A;
A113C, A339C, A120C, Q428C, T160F, I177L, D183P, T59A, and T174A;
A113C, A339C, A120C, Q426C, T160F, I177L, A185P, T59A, and T174A;
A113C, A339C, A120C, Q428C, T160F, I177L, A185P, T59A, and T174A;
A113C, A339C, A120C, Q426C, T160F, I177L, D186P, T59A, and T174A; or
A113C, A339C, A120C, Q428C, T160F, I177L, D186P, T59A, and T174A;
to stabilize 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.

### Claim 16 (depends on 1)

The nucleic acid molecule of claim 1, wherein:
the N-terminal residue of the F2 polypeptide is one of MPV F positions 8-30;
the C-terminal residue of the F2 polypeptide is one of MPV F positions 90-102;
the N-terminal residue of the F1 ectodomain is one of MPV F positions 103-130; and/or
the C-terminal residue of the F1 ectodomain is one of MPV F positions 470-550, and
wherein the positions correspond to a reference MPV F sequence set forth as SEQ ID NO: 7.

### Claim 17 (depends on 1)

The nucleic acid molecule of claim 1, wherein the recombinant MPV F protein further comprises a deletion of residues 103-123 to remove a fusion peptide of the F1 ectodomain, wherein the amino acid positions correspond to a reference MPV F sequence set forth as SEQ ID NO: 7.

### Claim 18 (depends on 1)

The nucleic acid molecule of claim 1, wherein the F2 polypeptide and/or the F1 ectodomain comprises an amino acid sequence at least 80% identical to a corresponding F2 polypeptide or F1 ectodomain from a human A1, A2, B1, or B2 strain of MPV.

### Claim 19 (depends on 1)

The nucleic acid molecule of claim 1, wherein the recombinant MPV F protein is a human subgroup A or human subgroup B MPV F protein.

### Claim 20 (depends on 1)

The nucleic acid molecule of claim 1, wherein the native MPV F protein sequence comprises the amino acid sequence set forth as SEQ ID NO: 1 (NL/1/00), SEQ ID NO: 2 (CAN97-83), SEQ ID NO: 3 (NL/17/00), SEQ ID NO: 4 (NCL174), SEQ ID NO: 5 (NL/1/99), SEQ ID NO: 6 (NDL00-1), or SEQ ID NO: 7 (CAN98-75).

### Claim 21 (depends on 1)

The nucleic acid molecule of claim 1, wherein the recombinant MPV F protein comprises the amino acid sequence set forth as any one of SEQ ID NOs: 12-13, 15, 80-81, 101-149, or 157-184, or an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 12-13, 15, 80-81, 101-149, or 157-184.

### Claim 22 (depends on 1)

The nucleic acid molecule of claim 1, wherein the recombinant MPV F protein further comprises one or more amino acid substitutions that introduce an enhanced furin cleavage site between the F2 polypeptide and the F1 ectodomain.

### Claim 23 (depends on 22)

The nucleic acid molecule of claim 22, wherein the enhanced furin cleavage site comprises the amino acid sequence set forth as RRRRRR (SEQ ID NO: 9).

### Claim 24 (depends on 1)

The nucleic acid molecule of claim 1, wherein the recombinant MPV F protein is a single chain MPV F protein and further comprises one or more amino acid substitutions to remove a protease cleavage site between the F2 polypeptide and the F1 ectodomain, and the F2 polypeptide and F1 ectodomain are linked by a heterologous peptide linker, or are directly linked.

### Claim 25 (depends on 24)

The nucleic acid molecule of claim 24, wherein the heterologous peptide linker joins MPV F positions 97 and 107, 98 and 103, 98 and 107, 100 and 103, or 101 and 103, wherein the amino acid positions correspond to a reference MPV F sequence set forth as SEQ ID NO: 7.

### Claim 26 (depends on 1)

The nucleic acid molecule of claim 1, wherein the recombinant MPV F protein forms a trimer of recombinant MPV F proteins stabilized in the prefusion conformation.

### Claim 27 (depends on 1)

The nucleic acid molecule of claim 1, wherein a C-terminal residue of the F1 ectodomain is linked to a trimerization domain by a peptide linker or is directly linked.

### Claim 28 (depends on 27)

The nucleic acid molecule of claim 27, wherein the trimerization domain is a foldon domain.

### Claim 29 (depends on 28)

The nucleic acid molecule of claim 28, wherein the foldon domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 33.

### Claim 30 (depends on 27)

The nucleic acid molecule of claim 27, wherein the recombinant MPV F protein comprises or consists of an amino acid sequence at least 90% identical to SEQ ID NO: 192.

### Claim 31 (depends on 30)

The nucleic acid molecule of claim 30, wherein the recombinant MPV F protein comprises or consists of the amino acid sequence set forth as SEQ ID NO: 192.

### Claim 32 (depends on 1)

The nucleic acid molecule of claim 1, wherein the C-terminus of the F1 ectodomain of the MPV F protein is linked to a transmembrane domain by a peptide linker, or is directly linked.

### Claim 33 (depends on 1)

The nucleic acid molecule of claim 1, wherein the C-terminus of the F1 ectodomain of the MPV F protein is linked to a self-assembling protein nanoparticle subunit by a peptide linker, or is directly linked.

### Claim 34 (depends on 33)

The nucleic acid molecule of claim 33, wherein the self-assembling protein nanoparticle subunit is a ferritin nanoparticle subunit or a lumazine synthase nanoparticle subunit.

### Claim 35 (depends on 1)

The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is an RNA molecule.

### Claim 36 (depends on 1)

An expression vector comprising the nucleic acid molecule of claim 1, operably linked to a promoter.

### Claim 37 (depends on 36)

The expression vector of claim 36, wherein the vector is a viral vector.