Recombinant protein expression using a hybrid CHEF1 promoter
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Publication Number
US-9297024-B2
Publication Date
2016-03-29
Expiration Date
Abstract
The invention provides expression vectors and host cells for high-level expression of recombinant proteins. The expression vectors comprise Chinese hamster ovary elongation factor 1-α (CHEF1) transcriptional regulatory DNA elements and a cytomegalovirus (CMV) promoter and/or a human adenovirus tripartite leader (AdTPL) sequence. The invention achieves increased protein expression and better productivity of host cells compared to previously described expression systems.
Core Innovation
The invention relates to recombinant protein expression using an expression vector that includes Chinese Hamster Elongation Factor-1α (CHEF1) transcriptional regulatory DNA combined with a cytomegalovirus (CMV) promoter and/or an adenovirus tripartite leader (AdTPL) sequence. The CHEF1 transcriptional regulatory DNA comprises defined CHEF1 regions identified as Sequence ID NOs 1-3.
Host cells, including CHO cells and non-hamster mammalian cells, including human cells, transformed with the CHEF1-CMV and/or CHEF1-AdTPL hybrid systems show synergistically increased recombinant protein expression and improved productivity relative to systems using CHEF1-only or CMV-only regulatory arrangements. The document describes expression vector constructs that combine the CHEF1 transcriptional regulatory DNA with CMV promoter and/or AdTPL, including constructs identified as pDEF85 (CHEF1-CMV) and pDEF86 (CHEF1-CMV-AdTPL).
The document provides examples in which CHEF1-CMV and CHEF1-CMV-AdTPL constructs drive expression of GP1 (Fc fusion) and MAb1 (IgG1) in CHO stable pools and clonal lines. Reported results include increased titers and specific productivity, and a temporal shift in peak expression toward later stationary-phase expression for CHEF1-CMV constructs. The document further indicates possible additional benefit for specific productivity from inclusion of AdTPL in the CHEF1-CMV-AdTPL configuration.
Claims Coverage
The provided independent claims cover expression vectors defined by CHEF1 transcriptional regulatory DNA and inclusion of a CMV promoter and/or an adenovirus tripartite leader (AdTPL) sequence, with the CHEF1 transcriptional regulatory DNA specifically limited to defined Sequence ID NOs. The claim set includes 3 main inventive feature groupings across the independent claims, centered on vector regulatory DNA composition, specific CHEF1 Sequence ID selection, and optional downstream refinements in the family that relate to host cells and encoded protein expression components.
Hybrid CHEF1 transcriptional regulatory DNA with CMV promoter and/or AdTPL
An expression vector comprising Chinese Hamster Elongation Factor-1α (CHEF1) transcriptional regulatory DNA and a cytomegalovirus (CMV) promoter and/or an adenovirus tripartite leader (AdTPL) sequence.
CHEF1 transcriptional regulatory DNA defined as Sequence ID NO: 1
The expression vector of Hybrid CHEF1 transcriptional regulatory DNA with CMV promoter and/or AdTPL wherein the CHEF1 transcriptional regulatory DNA comprises Sequence ID NO: 1.
CHEF1 transcriptional regulatory DNA defined as Sequence ID NO: 2
The expression vector of Hybrid CHEF1 transcriptional regulatory DNA with CMV promoter and/or AdTPL wherein the CHEF1 transcriptional regulatory DNA comprises Sequence ID NO: 2.
CHEF1 transcriptional regulatory DNA defined as Sequence ID NO: 3
The expression vector of Hybrid CHEF1 transcriptional regulatory DNA with CMV promoter and/or AdTPL wherein the CHEF1 transcriptional regulatory DNA comprises Sequence ID NO: 3.
Across the independent claims, the inventive coverage focuses on expression vectors that combine CHEF1 transcriptional regulatory DNA with a CMV promoter and/or an AdTPL sequence, with the CHEF1 transcriptional regulatory DNA limited to specific defined Sequence ID NOs (1, 2, or 3).
Stated Advantages
Synergistically increased recombinant protein expression versus CHEF1-only or CMV-only systems.
Improved productivity versus CHEF1-only or CMV-only systems.
Increased titers for recombinant protein expression in described examples.
Improved specific productivity in described examples.
Temporal shift to later stationary-phase peak expression for CHEF1-CMV constructs.
Possible further benefit for specific productivity from inclusion of AdTPL in CHEF1-CMV-AdTPL constructs.
Documented Applications
Recombinant protein expression in transformed host cells, including CHO cells and non-hamster mammalian cells, including human cells.
Expression of GP1 (Fc fusion) in CHO stable pools and clonal lines.
Expression of MAb1 (IgG1) in CHO stable pools and clonal lines.
Fed-batch production models assessing specific productivity, cell viability, and stationary-phase expression timing.
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