Cassette encoding a FPV/brachyury fusion protein
Inventors
Li, Zengji • Delcayre, Alain • Rountree, Ryan
Assignees
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Publication Number
US-11753652-B2
Publication Date
2023-09-12
Expiration Date
Abstract
The present invention relates to one or more promoters and/or expression cassettes that can be used for enhancing expression of a heterologous gene, such as Brachury. In particular, the one or more promoters and/or expression cassettes enhance expression of heterologous genes as part of a viral vector, such as a poxvirus.
Core Innovation
The patent addresses insufficient expression of the tumor antigen Brachyury using known vaccinia promoters, including PrS and VV-40k. It states that poxviruses promoters and expression cassettes are used to enhance expression of Brachyury, because prior promoter choices do not provide adequate Brachyury expression for the intended application.
The disclosure identifies specific FPV promoter sequences, including SEQ ID NOs 1-10 and 77, that increase Brachyury expression. These promoter sequences are connected to expression cassettes in recombinant poxviruses that deliver the Brachyury antigen expression cassette.
The patent further describes expression cassettes encoding fusion protein constructs that include an FPV 088 antigen and the Brachyury antigen. It also includes nucleic acid and protein variants for Brachyury and related components by sequence identifier ranges, and describes recombinant virus and vaccine compositions driven by FPV promoter sequences, including a clinical-stage FPV-mBN345B variant with TRICOM and a Brachyury cassette driven by an I3+15aa promoter.
Supporting in-cell data are reported using western blot and flow cytometry (FACS) comparing multiple recombinant FPV strains and promoter constructs. The disclosure states that Brachyury expression detection and relative expression levels are assessed in human dendritic cells (DCs) and CMMT cells, and that the FPV promoter sequences improve Brachyury expression relative to promoter controls and contexts described in the patent.
Claims Coverage
The partial content includes three independent claims, directed to (i) an expression cassette encoding a fusion protein comprising FPV 088 antigen and a Brachyury antigen, (ii) an expression cassette comprising SEQ ID NO:72, and (iii) an expression cassette comprising SEQ ID NO:73. Across these independent claims, the inventive coverage centers on defined expression cassette sequence content and, for one claim, a fusion protein architecture including an FPV 088 antigen with Brachyury.
Expression cassette encoding a fusion protein comprising FPV 088 antigen and a Brachyury antigen
An expression cassette comprising the nucleic acid sequence of SEQ ID NO:6 operably linked to a nucleic acid sequence encoding a Brachyury antigen, wherein the expression cassette encodes a fusion protein comprising a FPV 088 antigen and said Brachyury antigen.
Expression cassette comprising SEQ ID NO:72
An expression cassette comprising the nucleic acid sequence of SEQ ID NO:72.
Expression cassette comprising SEQ ID NO:73
An expression cassette comprising the nucleic acid sequence of SEQ ID NO:73.
Overall, the independent claims define specific expression cassette sequence compositions, including a fusion construct that links SEQ ID NO:6 with a Brachyury antigen to encode a fusion protein containing FPV 088 antigen, and two further expression cassettes defined solely by inclusion of SEQ ID NO:72 or SEQ ID NO:73.
Stated Advantages
Increased Brachyury expression using specific FPV promoter sequences (SEQ ID NOs 1-10 and 77), including in recombinant poxviruses.
Documented Applications
Recombinant poxviruses and related vaccine compositions that deliver expression cassettes to achieve Brachyury antigen expression, assessed in human dendritic cells (DCs) and CMMT cells.
A clinical-stage FPV-mBN345B variant with TRICOM and a Brachyury cassette driven by an I3+15aa promoter, including removal of a drug selection cassette.
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