Modified cytomegalovirus proteins and stabilized complexes

Patent metadata

Field	Value
Publication number	US-11932669-B2
Application number	US-202117223207-A
Country	US
Kind code	B2
Filing date	Apr 6, 2021
Priority date	Oct 17, 2018
Publication date	Mar 19, 2024
Grant date	Mar 19, 2024

Abstract

Described are mutant human cytomegalovirus (HCMV) pentamer complex polypeptides, methods of making them, and their use in HCMV protein complexes and compositions. In particular, the use of the modified HCMV polypeptides to stabilize HCMV complexes or unmask a pentamer epitope is described.

First claim

The invention claimed is:

1. A complex comprising one or more mutant Human Cytomegalovirus (HCMV) polypeptides, wherein the one or more mutant HCMV polypeptides are:
(a) a pUL128 polypeptide, or complex-forming fragment thereof, that has cysteine (C) at residue 142, numbered with respect to SEQ ID NO: 13;
a gL polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 140, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, and has a cysteine (C) at residue 150, numbered with respect to SEQ ID NO: 7; and
a pUL130 polypeptide, or complex-forming fragment thereof, that has a cysteine (C) at residue 64, numbered with respect to SEQ ID NO: 17, and a cysteine (C) at residue 95, numbered with respect to SEQ ID NO: 17;

(b) a pUL128 polypeptide, or complex-forming fragment thereof, that has cysteine (C) at residue 142, numbered with respect to SEQ ID NO: 13;
a gL polypeptide, or complex-forming fragment thereof, that has a cysteine (C) at residue 150, numbered with respect to SEQ ID NO: 7; and
a pUL130 polypeptide, or complex-forming fragment thereof, that has a cysteine (C) at residue 64, numbered with respect to SEQ ID NO: 17, and a cysteine (C) at residue 95, numbered with respect to SEQ ID NO: 17;

(c) a pUL128 polypeptide, or complex-forming fragment thereof, that has cysteine (C) at residue 142, numbered with respect to SEQ ID NO: 13;
a gL polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 145, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, and has a cysteine (C) at residue 150, numbered with respect to SEQ ID NO: 7; and
a pUL130 polypeptide, or complex-forming fragment thereof, that has a cysteine (C) at residue 64, numbered with respect to SEQ ID NO: 17, and a cysteine (C) at residue 95, numbered with respect to SEQ ID NO: 17;

(d) a pUL128 polypeptide, or complex-forming fragment thereof, that has cysteine
(C) at residue 142, numbered with respect to SEQ ID NO: 13;

a gL polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 224, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, and has a cysteine (C) at residue 150, numbered with respect to SEQ ID NO: 7; and
a pUL130 polypeptide, or complex-forming fragment thereof, that has a cysteine (C) at residue 64, numbered with respect to SEQ ID NO: 17, and a cysteine (C) at residue 95, numbered with respect to SEQ ID NO: 17;
(e) a pUL128 polypeptide, or complex-forming fragment thereof, that has cysteine
(C) at residue 142, numbered with respect to SEQ ID NO: 13;

a gL polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 224, numbered with respect to the wild-type Sequence of SEQ ID NO: 7; and
a pUL130 polypeptide, or complex-forming fragment thereof, that has a cysteine (C) at residue 95, numbered with respect to SEQ ID NO: 17;
(f) a pUL128 polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 77, numbered with respect to the wild-type Sequence of SEQ ID NO: 13, and has a cavity filling mutant at residue 103, numbered with respect to the wild-type Sequence of SEQ ID NO: 13; and
a gL polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 140, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, and has a cavity filling mutant at residue 145, numbered with respect to the wild-type Sequence of SEQ ID NO: 7;
(g) a pUL128 polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 77, numbered with respect to the wild-type Sequence of SEQ ID NO: 13, and has a cavity filling mutant at residue 103, numbered with respect to the wild-type Sequence of SEQ ID NO: 13; and
a gL polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 140, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, has a cavity filling mutant at residue 145, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, and has a cavity filling mutant at residue 224, numbered with respect to the wild-type Sequence of SEQ ID NO: 7;
(h) a gL polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 140, numbered with respect to the wild-type Sequence of SEQ ID NO: 7; and
a pUL131 polypeptide, or complex-forming fragment thereof, that has a repacking hydrophobic mutant at residue 52, numbered with respect to the wild-type Sequence of SEQ ID NO: 21, and has a repacking hydrophobic mutant at residue 67, numbered with respect to the wild-type Sequence of SEQ ID NO: 21;
(i) a pUL131 polypeptide, or complex-forming fragment thereof, that has a cavity filling mutant at residue 86, numbered with respect to the wild-type Sequence of SEQ ID NO: 21; or
(j) a pUL128 polypeptide, or complex-forming fragment thereof, that has a cysteine
(C) at residue 83, numbered with respect to SEQ ID NO: 13; and

a pUL130 polypeptide, or complex-forming fragment thereof, that has a cysteine
(C) at residue 167, numbered with respect to SEQ ID NO: 17.

2. The complex of claim 1 , wherein the cavity filling mutant at residue 140, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, is tryptophan (W), phenylalanine (F), tyrosine (Y), valine (V), isoleucine (I), or leucine (L).

3. The complex of claim 1 , wherein the cavity filling mutant at residue 145, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, is tryptophan (W), phenylalanine (F), tyrosine (Y), valine (V), isoleucine (I), or leucine (L).

4. The complex of claim 1 , wherein the cavity filling mutant at residue 224, numbered with respect to the wild-type Sequence of SEQ ID NO: 7, is tryptophan (W), phenylalanine (F), tyrosine (Y), valine (V), isoleucine (I), or leucine (L).

5. The complex of claim 1 , wherein the cavity filling mutant at residue 77, numbered with respect to the wild-type Sequence of SEQ ID NO: 13, is tryptophan (W), phenylalanine (F), tyrosine (Y), isoleucine (I), or leucine (L).

6. The complex of claim 1 , wherein the cavity filling mutant at residue 103, numbered with respect to the wild-type Sequence of SEQ ID NO: 13, is tryptophan (W), phenylalanine (F), tyrosine (Y), valine (V), or isoleucine (I).

7. The complex of claim 1 , wherein the repacking hydrophobic mutant at residue 52, numbered with respect to the wild-type Sequence of SEQ ID NO: 21, is tryptophan (W), phenylalanine (F), methionine (M), cysteine (C), alanine (A), leucine (L), isoleucine (I), valine (V) or proline (P).

8. The complex of claim 1 , wherein the repacking hydrophobic mutant at residue 67, numbered with respect to the wild-type Sequence of SEQ ID NO: 21, is tryptophan (W), phenylalanine (F), methionine (M), cysteine (C), leucine (L), isoleucine (I), valine (V) or proline (P).

9. The complex of claim 1 , wherein the cavity filling mutant at residue 86, numbered with respect to the wild-type Sequence of SEQ ID NO: 21, is tryptophan (W), phenylalanine (F), tyrosine (Y), valine (V), isoleucine (I), or leucine (L).

10. The complex of claim 1 , wherein the complex has an increased thermostability as compared to a control complex in the same conditions.

11. The complex of claim 1 , wherein the complex is an HCMV pentamer complex comprising one or more mutant HCMV polypeptides.

12. An immunogenic composition comprising the complex of claim 1 .

13. An isolated nucleic acid molecule comprising one or more operably linked polynucleotide sequences that encode the complex of claim 1 .

14. An expression vector comprising the isolated nucleic acid molecule of claim 1

Assignees

• Glaxosmithkline Biologicals Sa

Inventors

• Malito Enrico
• Bottomley Matthew James
• Carfi Andrea
• Chandramouli Sumana
• Luisi Kate

Classifications

• A61K39/245

  Herpetoviridae, e.g. herpes simplex virus · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C07K14/045 Primary

  Cytomegalovirus · CPC title

• A61K39/12 Primary

  Viral antigens · CPC title

• C07K14/005

  from viruses · CPC title