Conformationally stabilized RSV pre-fusion F proteins
Inventors
Marshall, Christopher Patrick • McLellan, Jason Scott • Alff, Peter Joseph • Bertuccioli, Claudio • Mariani, Roberto
Assignees
Calder Biosciences Inc • US Department of Health and Human Services
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Publication Number
US-10125172-B2
Publication Date
2018-11-13
Expiration Date
2034-07-24
Abstract
In some embodiments, the present invention provides respiratory syncytial virus (RSV) F proteins, polypeptides and protein complexes that comprise one or more cross-links to stabilize the protein, polypeptide or protein complex in its pre-fusion conformation. In some embodiments the present invention provides RSV F proteins, polypeptides and protein complexes comprising one or more mutations to facilitate such cross-linking. In some embodiments the present invention provides compositions comprising such proteins, polypeptides or protein complexes, including vaccine compositions, and methods of making and using the same.
Core Innovation
The present invention provides respiratory syncytial virus (RSV) F proteins, polypeptides and protein complexes that are stabilized in their pre-fusion conformation by introducing one or more cross-links, such as targeted di-tyrosine cross-links. The invention includes RSV F proteins comprising mutations to facilitate cross-linking, specifically the addition of point mutations to tyrosine at specific amino acid residues. Such stabilization aims to lock or stabilize the RSV F protein in the pre-fusion conformation, which is important for inducing potent neutralizing antibodies.
The problem being solved originates from the instability of the RSV F protein in its pre-fusion conformation, which readily transitions to a post-fusion form, limiting its usefulness as a vaccine immunogen. Current prophylactic treatment with Synagis is only partially effective and costly. The development of a stabilized RSV F protein locked in the pre-fusion conformation could provide a more effective vaccine immunogen and aid the development of antibodies for diagnostic and therapeutic purposes.
The invention identifies specific locations within the RSV F protein for cross-linking and provides RSV F polypeptides with engineered point mutations to tyrosine to enable di-tyrosine cross-links. The cross-links may be intra- or intermolecular, inter-protomer or intra-protomer, and can be used singly or in combinations. The invention includes compositions comprising such stabilized proteins and methods of making and using them as vaccines or for eliciting neutralizing antibodies.
Claims Coverage
The patent includes 15 claims focusing on the stabilization of RSV F proteins with tyrosine mutations and di-tyrosine cross-links. The main inventive features relate to specific mutations, stabilization by cross-linking, binding properties, additional modifications, nucleic acids encoding the proteins, and compositions comprising them.
Point mutation to tyrosine at position 226
An RSV F polypeptide, protein or protein complex comprising a point mutation to tyrosine at amino acid position 226 or an equivalent position.
Stabilization by di-tyrosine cross-links
The RSV F polypeptide, protein or protein complex is stabilized in the prefusion conformation by one or more di-tyrosine cross-links.
Specific di-tyrosine cross-link between positions 226 and 198
The protein comprises a di-tyrosine cross-link between the tyrosine at position 226 and tyrosine at position 198, or equivalent positions.
Derivation from RSV type A or B
The RSV F polypeptide, protein or protein complex is derived from RSV type A or RSV type B.
Capability to bind antibody recognizing antigenic site ø
The RSV F polypeptide, protein or protein complex is capable of binding to an antibody that recognizes antigenic site ø.
Compositions comprising specific sequences
The RSV F polypeptide, protein or protein complex comprises amino acid residues 1-513 of SEQ ID NOs: 15, 27, 28, or 32.
Inclusion of additional artificially introduced cross-links
The protein further comprises one or more additional artificially introduced cross-links, such as disulfide bonds.
Presence of point mutations to cysteine
The polypeptide, protein or protein complex comprises one or more point mutations to cysteine to facilitate disulfide bonds.
Cavity-filling hydrophobic amino acid substitutions
The protein further comprises one or more cavity-filling hydrophobic amino acid substitutions.
Inclusion of a trimerization domain
The RSV F polypeptide, protein or protein complex further comprises a trimerization domain, specifically a foldon domain.
Nucleic acid encoding the RSV F protein with tyrosine mutation
A nucleic acid molecule encoding an RSV F polypeptide, protein or protein complex comprising a tyrosine mutation at amino acid position 226 or equivalent.
Compositions comprising the stabilized RSV F protein
Pharmaceutical or vaccine compositions comprising the RSV F polypeptide, protein or protein complex having the tyrosine mutation at position 226 or equivalents.
The claims encompass RSV F proteins and polypeptides with specific tyrosine mutations that enable di-tyrosine cross-link stabilization in the prefusion conformation, including particular cross-link pairs, additional structural modifications such as disulfide bonds and cavity-filling mutations, trimerization domains, encoding nucleic acids, and compositions comprising these engineered proteins for use in vaccines or therapeutics.
Stated Advantages
Stabilization of the RSV F protein in the pre-fusion conformation enhances its usefulness as a vaccine immunogen.
Di-tyrosine cross-linking provides stable, irreversible bonds that maintain protein structure without leading to aggregation during expression or storage.
Stabilized RSV F proteins are capable of binding to potent, prefusion-specific neutralizing antibodies and can elicit protective immune responses.
The cross-linking strategy is scalable and compatible with manufacturing processes.
Documented Applications
Use of the stabilized RSV F proteins, polypeptides, and complexes as vaccine immunogens to elicit RSV-specific neutralizing antibodies and protective immune responses in subjects.
Use as therapeutic agents or components of therapeutic compositions to treat or prevent RSV infection.
Use as targets for generation of antibodies, including monoclonal, chimeric, humanized, or fully human antibodies for therapy or research.
Use as diagnostic tools and research reagents to analyze RSV F antibody binding and specificity.
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