FhuD2 fusion proteins for the outer membrane vesicle (OMV) delivery of heterologous polypetides and immunogenic compositions thereof
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Publication Number
US-12054521-B2
Publication Date
2024-08-06
Expiration Date
Abstract
There are disclosed fusion proteins comprising the bacterial protein FhuD2 and one or more copies of a heterologous polypeptide, polynucleotides and expression vectors encoding the fusion proteins and bacterial outer membrane vesicles containing them. Other aspects of the invention regard immunogenic compositions comprising the outer membrane vesicles and their use in the prevention or treatment of tumors.
Core Innovation
A bacterial outer membrane vesicle is provided that comprises a fusion protein displayed on the OMV surface. The fusion protein includes the bacterial protein FhuD2 fused to one or more copies of a heterologous polypeptide, where the heterologous polypeptide is linked to the carboxyl terminus of FhuD2. FhuD2 carries an acylated N-terminal residue, and the resulting fusion protein is expressed on the OMV surface.
The OMV surface display is tied to an acylated N-terminal residue on FhuD2, and in refinements to an acylated N-terminal cysteine residue resulting from cleavage of a lipoprotein leader sequence in an immature precursor form of the fusion protein. The number of heterologous polypeptide copies can be constrained, and the heterologous polypeptide can be linked to tumor-associated antigens with optional specified immune epitope types.
Methods are also provided for preparing bacterial OMVs that produce the same surface-displayed fusion architecture. The method includes expressing, on the surface of a Gram-negative bacterium, a fusion protein in which FhuD2 is linked at its carboxyl terminus to one or more copies of a heterologous polypeptide, where FhuD2 carries an acylated N-terminal residue, and then isolating the outer membrane vesicles from the bacterial culture.
Claims Coverage
The provided material includes two independent claims: one directed to an isolated bacterial OMV with a specific surface-displayed FhuD2 fusion protein, and one directed to a method of preparing such OMVs by expressing the surface fusion protein in a Gram-negative bacterium and isolating OMVs. Across the independent claims, the main inventive elements are the FhuD2-carboxyl-terminal fusion to heterologous polypeptides, the presence of an acylated N-terminal residue carried by FhuD2, and surface expression on the OMV, with preparation requiring surface expression in a Gram-negative bacterium followed by OMV isolation.
Surface-displayed FhuD2-carboxyl-terminal heterologous polypeptide fusion on OMVs
The isolated bacterial outer membrane vesicle comprises a fusion protein wherein the bacterial protein FhuD2 is fused to one or more copies of a heterologous polypeptide and the heterologous polypeptide is linked to the carboxyl terminus of FhuD2.
Acylated N-terminal residue carried by FhuD2
The fusion protein includes that FhuD2 carries an acylated N-terminal residue.
Fusion protein expressed on the OMV surface
The fusion protein is expressed on the OMV surface.
Expressing surface fusion protein in a Gram-negative bacterium and isolating OMVs
A method of preparing a bacterial outer membrane vesicle comprising a fusion protein wherein FhuD2 is fused to one or more copies of a heterologous polypeptide linked to the carboxyl terminus of FhuD2, FhuD2 carries an acylated N-terminal residue, and the fusion protein is expressed on the OMV surface, comprising expressing on the surface of a Gram-negative bacterium the fusion protein and isolating the outer membrane vesicle from the bacterial culture.
Overall, the claim coverage centers on OMVs and OMV preparation in which FhuD2 is used as the carrier to create a surface-expressed fusion protein that links a heterologous polypeptide to the FhuD2 carboxyl terminus while maintaining an acylated N-terminal residue on FhuD2.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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