JPWO2021163481A5

JPWO2021163481A5 -

Info

Publication number: JPWO2021163481A5
Application number: JP2022549070A
Authority: JP
Publication date: 2024-02-19

Claims

The polypeptide is
(a) At least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence of SEQ ID NO: 1 (I3-01 wild type) , 98%, or 99% identical amino acid sequence to SEQ ID NO: 1, with the following mutations: F32Y, H37D/E/K/N/Q/R, F43Q, F168D/E/K/N/ Q/R/S/T/Y, K169D/E/N/Q, L173D/E/N/Q/S, A174S, S179D/E, K183D/E, and/or T185D/E/K/N/Q an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or all 10 of /S are present in the polypeptide;
(b) At least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 2 (O43-38 tetramer wild type) , 97%, 98%, or 99% identical amino acid sequence to SEQ ID NO: 2, the following mutations, M138D/E/K/N/Q/R/S/T, L139D/N/S , A141S, V142R/T, A143S, N146D/E/K/R, R147N, H172D/E/K/N/Q, and/or 1, 2, 3, 4, 5, 6 of E173D/K, an amino acid sequence of which 7, 8, or all 9 are present in said polypeptide;
(c) At least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 3 (O43-38 trimer wild type) , 97%, 98%, or 99% identical amino acid sequence to SEQ ID NO: 3, with the following mutations: R17D/E/K/N/Q/S/T, N19D/E, S20D/E /K/N, V21D/T, V22D/E/Q/S/T, L23D/E/K/N/Q/R/S, A26S, K27N/Q, A30S V31N/S/T, F32R/Y, L33D/E/K/N/Q/R/S/T, H37D/E/K/N/R, F43Q, W167D/E/K/N/Q/R/S/T/Y, F168D/E/ K/N/Q/R/S/T/Y, K169D/E/N, L173D/E/N/Q/R/S, A174S, S179D/E/K/N/Q/R, and/or K183D /E/N/Q of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or an amino acid sequence, all 21 of which are present in said polypeptide;
(d) the amino acid sequence of SEQ ID NO: 4 (I53_dn5A wild type) and at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%; % or 99% identical amino acid sequence to SEQ ID NO: 4, the following mutations: R17T, W18D/E/K/N/Q/R/S/T/Y, N19E, E21D, L28D/ E/K/N/Q/R/S/T/Y, L31D/E/K/N/Q/S/T, K32D/E/N/Q, T118D/E/N/Q/S, L120D/ 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of E/K/N/Q/R/S/T and/or T121D/E/K/N/S or (e) at least 75%, 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence of SEQ ID NO: 5 or 6 (hMPV wild type), all of which are present in said polypeptide. %, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequence to SEQ ID NO: 5 or 6, with the following mutations: A107D, V112R, T114E, V118R, and /or comprises or consists of an amino acid sequence in which 1, 2, 3, 4, or all 5 of G264D are present in said polypeptide;
The residues in parentheses are optional and may be present or absent in whole or in part in the polypeptide.

The polypeptide has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of SEQ ID NO: 1 (I3-01 wild type), Contains or consists of an amino acid sequence 97%, 98%, or 99% identical to SEQ ID NO: 1, with the following mutations: F32Y, H37D/E/K/N/Q/R, F43Q, F168D /E/K/N/Q/R/S/T/Y, K169D/E/N/Q, L173D/E/N/Q/S, A174S, S179D/E, K183D/E, and/or T185D/ all 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of E/K/N/Q/S are present in said polypeptide; optionally,
The polypeptide has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NOs: 7-14. , or comprises or consists of a 99% identical amino acid sequence, or
SEQ ID NO: 2 (O43-38 tetramer wild type) and at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , or a polypeptide comprising or consisting of a 99% identical amino acid sequence, with respect to SEQ ID NO: 2, the following mutations, M138D/E/K/N/Q/R/S/T, L139D 1, 2, 3, 4 of /N/S, A141S, V142R/T, A143S, N146D/E/K/R, R147N, H172D/E/K/N/Q, and/or E173D/K, 5, 6, 7, 8, or all 9 are present in said polypeptide, optionally;
The polypeptide has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NOs: 24-25. , or comprises or consists of a 99% identical amino acid sequence, or
The polypeptide has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence of SEQ ID NO: 3 (O43-38 trimer wild type), Contains or consists of an amino acid sequence 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3, with the following mutations: R17D/E/K/N/Q/S/T; N19D/E, S20D/E/K/N, V21D/T, V22D/E/Q/S/T, L23D/E/K/N/Q/R/S, A26S, K27N/Q, A30S V31N/S /T, F32R/Y, L33D/E/K/N/Q/R/S/T, H37D/E/K/N/R, F43Q, W167D/E/K/N/Q/R/S/T /Y, F168D/E/K/N/Q/R/S/T/Y, K169D/E/N, L173D/E/N/Q/R/S, A174S, S179D/E/K/N/Q /R, and/or K183D/E/N/Q 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, all 18, 19, 20, or 21 are present in said polypeptide, optionally;
The polypeptide has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NOs: 26-28. or comprises or consists of a 99% identical amino acid sequence, or
The polypeptide has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NO: 4 (I53_dn5A). % or 99% identical amino acid sequence, or the following mutations to SEQ ID NO: 4, R17T, W18D/E/K/N/Q/R/S/T/Y, N19E , E21D, L28D/E/K/N/Q/R/S/T/Y, L31D/E/K/N/Q/S/T, K32D/E/N/Q, T118D/E/N/Q 1, 2, 3, 4, 5, 6, 7, 8 of /S, L120D/E/K/N/Q/R/S/T, and/or T121D/E/K/N/S, 9, or all 10 are present in said polypeptide , and optionally,
The polypeptide has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NO: 15-23. or comprises or consists of a 99% identical amino acid sequence, or
The polypeptide has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence of SEQ ID NO: 5 (hMPV wild type) or 6 (hMPV or 115-BV). , 95%, 96%, 97%, 98%, or 99% identical amino acid sequence, or consists of the following mutations to SEQ ID NO: 5 or 6, A107D, V112R, T114E, V118R, and/or all 1, 2, 3, 4, or 5 of G264D are present in said polypeptide, and optionally,
The polypeptide has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NO: 5 or 6. , or a set of mutations to SEQ ID NO: 5 or 6 comprising or consisting of a 99% identical amino acid sequence, wherein said polypeptide is selected from the group consisting of:
(a) T114E+V118R
(b) A107D+V112R+T114E+V118R
(c) A107D+V112R
(d) A107D+V112R+T114E+V118R; and (e) A107D+V112R+T114E+V118R+G264D; , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences; The residues are optional and may or may not be present in whole or in part, optionally
2. The polypeptide of claim 1 , wherein some or all of the residues within parentheses are absent or present .

(a) the polypeptide according to claim 2;
(b) a second functional polypeptide; optionally, a fusion protein comprising:
said second functional polypeptide comprises an immunogenic portion of a polypeptide antigen, and optionally,
The fusion protein has at least 75%, 80%, 85%, 90%, 91%, 92%, 93 with the amino acid sequence of SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 44, or 46. %, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequence, residues in parentheses are optional and may be present in whole or in part. A fusion protein that can be used or not.

Nanoparticles comprising a plurality of polypeptides or fusion proteins according to any one of claims 1 to 3 .

A composition comprising a plurality of nanoparticles according to claim 4 .

A nucleic acid encoding a polypeptide or fusion protein according to any one of claims 1 to 3 .

An expression vector comprising a nucleic acid according to claim 6 operably linked to appropriate control sequences.

A host cell comprising a polypeptide, fusion protein, nanoparticle, composition, nucleic acid, and/or expression vector according to any one of claims 1 to 7 .

A pharmaceutical composition comprising:
(a) a polypeptide, fusion protein, nanoparticle, composition, nucleic acid, expression vector, and/or host cell according to any one of claims 1 to 8 ; and (b) a pharmaceutically acceptable carrier. A pharmaceutical composition comprising .

A synthetic (“defatted”) nanoparticle comprising a potential transmembrane domain, wherein one or more of the hydrophobic amino acids of said potential transmembrane domain is replaced with a polar amino acid , and optionally,
It was confirmed that the amino acid substitution was within a 19-residue sliding window of transmembrane insertion potential (dG_ins), and that the window of dG_ins below +2.7 kcal/mol was a minimum value within +/−9 residues. , +2.7 kcal/mol cutoff is characteristic of the potential transmembrane domain of synthetic nanoparticles.

A synthetic nanoparticle comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 13.

Synthetic nanoparticles according to any one of claims 10 to 11 , wherein the synthetic nanoparticles are polypeptides, optionally comprising:
said synthetic nanoparticle comprises a signal peptide, and optionally,
the synthetic nanoparticle comprises a tag, optionally;
The synthetic nanoparticles include monocomponent or homomeric nanoparticles, optionally comprising:
Said synthetic nanoparticle comprises a variant I3-01 amino acid sequence, preferably
The synthetic nanoparticle comprises a polar amino acid substitution at position 25, 35, 171, 177, or 180, or a combination of any two or more of these positions, and optionally,
Said synthetic nanoparticle further comprises a secreted agent (“secreted agent”), preferably:
The secretory agent is
a) polypeptide;
b) payload;
c) an antigen displayed on the outside of said synthetic nanoparticle , optionally;
The polypeptide comprises an antigen or an immunogenic portion of an antigen, preferably
Said antigen or immunogenic part of an antigen is of viral origin, preferably
the virus is human metapneumovirus (hMPV), or
The synthetic nanoparticles include binary nanoparticles, preferably
the synthetic nanoparticle comprises a trimer, a tetramer, or a pentamer , or
the synthetic nanoparticles are selected from I53_dn5, O43-38, and I53-50 , or
The synthetic nanoparticle is I53_dn5, and the pentameric subunit I53_dn5A of the synthetic nanoparticle is at least one of the 16th, 29th, 116th, 118th, or 119th positions, or any of these positions. contains a polar amino acid substitution in a combination of two or more of; or
The synthetic nanoparticle is O43-38, and the tetrameric subunit O43-38tet of the synthetic nanoparticle is at position 29, 141, 19, 21, or 31, or any of these positions. Synthetic nanoparticles containing polar amino acid substitutions in combination of two or more.

A nucleic acid molecule encoding a synthetic nanoparticle according to any one of claims 1 to 12 , preferably comprising:
A nucleic acid molecule, wherein said polynucleotide is mRNA.

An expression vector comprising the nucleic acid molecule according to claim 13 .

A cell comprising the nucleic acid molecule according to claim 13 and/or the expression vector according to claim 14 .

A secretion agent comprising administering or mixing the nucleic acid molecule and/or expression vector according to any one of claims 1 to 15 into a cell, and secreting the nanoparticle or the synthetic nanoparticle. How to deliver.

A vaccine comprising a nanoparticle, composition, pharmaceutical composition, synthetic nanoparticle, nucleic acid, expression vector, and/or cell according to any claim herein.

A method of vaccinating a subject against a virus, the method comprising administering to said subject a nanoparticle, composition, pharmaceutical composition, synthetic nanoparticle(s) or vaccine(s) as described herein. including, optionally,
(a) obtaining a nanoparticle, composition, pharmaceutical composition, synthetic nanoparticle, composition, or vaccine as described herein;
(b) administering to said subject a synthetic nanoparticle, composition, or vaccine described herein , optionally;
A method, wherein said administration induces an immune response in said subject such that said subject is protected from infection.

Polypeptide, fusion protein, nanoparticle, composition, synthetic nanoparticle(s), nucleic acid molecule(s), expression vector(s), cell(s) according to any of claims 1-18. , composition(s), or vaccine(s).

1. A computer-implemented method for designing secreted peptides, comprising:
generating a 3D structure of a protein of interest using a 19-residue sliding window of transmembrane insertion potential (dG_ins),
The window of dG_ins below +2.7 kcal/mol was confirmed to be a local minimum within +/-9 residues, and the +2.7 kcal/mol cutoff is characteristic of potential transmembrane domains. generating a 3D structure of a protein;
designing one or more peptide sequences based on the generated 3D structure and predicting mutations at each position within the domain, where the permissible residues include histidine and The final permissible residues are the amino acids D, E, K, R, Q, N, S, T, Y, and the side chains of other residues within the 8 angstrom shell. may adopt different rotamers ("repack" for those skilled in the art), but not be allowed to mutate to other residues ("design" for those skilled in the art), optionally
for each mutation or set of mutations, a score of the overall energy of the structure is generated;
(a) if the new score is higher than the original score by a threshold of 15 REU (dscore), the degreaser variant is discarded and not evaluated further;
(b) If the new score is within the acceptable range, but the change in dG_ins is less than +0.27 kcal/mol (ddG_ins), then said mutation placed at that position is rejected and is no longer allowed at that position; undergoes the mutation again, or (c) if the new score is within the tolerance range and said ddG_ins is greater than +0.27 kcal/mol, said mutation is accepted and said structure is optionally output and the mutation is A computer- implemented method in which metrics are written to a final output file.