Cell-associated secretion-enhancing fusion proteins
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Publication Number
US-11390899-B2
Publication Date
2022-07-19
Expiration Date
Abstract
Cell-associated secretion-enhancing fusion proteins are disclosed that comprise a target protein binding domain and a transmembrane retention domain. Co-expression in a host cell of a fusion protein and a target protein of interest that is temporarily bound by the fusion protein leads to an increased level of target protein secreted from the host cell. The fusion proteins are engineered to be retained with the producing host cell, thus eliminating a non-natural component from the extracellular media of the host cell and simplifying purification of the target protein. Nucleic acid molecules encoding such fusion proteins are also disclosed for use in expressing the fusion proteins in host cells, for use in restoring lost or diminished cell functions, and for use in treating diseases characterized by a lost or diminished cell function. Methods and compositions comprising fusion proteins of the invention are disclosed for use in enhancing the level of co-expressed target proteins secreted from host cells.
Core Innovation
The invention provides a cell-associated, secretion-enhancing (CASE) fusion protein comprising a target protein binding (TPB) domain and a transmembrane retention (TMR) domain, with an optional chaperone machinery peptide (CMP) domain. The CASE fusion protein is co-expressed in a host cell in culture with a target protein, and co-expression enhances the level of target protein secreted from the host cell into the culture media compared to expression of the target protein in the absence of the CASE fusion protein.
The disclosed constructs rely on selection of the TPB domain and the TMR domain, and optionally CMP, to achieve cell-associated secretion enhancement. The disclosure includes multiple CASE fusion protein variants with a Flag epitope and multiple linkers, and describes domain orientation and transmembrane designs including Type I, Type II, and Type III transmembrane regions as well as ER-resident-like behavior.
The TPB domain includes receptor/ligand-binding portions, truncated receptors fused to Fc as trap molecules, PDZ domains such as NHERF/NHERF family members, and Fc-binding domains or peptides such as HSV-1 gE, Protein A, Protein G, and FcBP. The disclosed TMR variants include VSV-G, KDELR/KDEL receptor-derived regions, truncated KDELR, p23, p24, LAMP2, LIMP2, CDM6PR, CNX, ERGIC53, gp84, and gp73.
The CMP domain includes J-domain fragments or analogs and other chaperone machinery peptide elements. The disclosure further notes that an ER-retention dilysine (KK) motif in the TMR abolishes secretion enhancement, and describes release behavior tied to secretory-pathway conditions, including pH changes along the secretory pathway, to avoid co-secretion of the CASE fusion protein into extracellular media while still enhancing target secretion.
Claims Coverage
The independent claim coverage centers on a CASE fusion protein architecture with three core structural components: TPB, TMR, and optional CMP. The claims repeatedly tie this architecture to a functional result, namely enhanced secretion of a co-expressed target protein into culture media relative to target expression alone.
Cell-associated secretion-enhancing fusion protein architecture
A cell-associated, secretion-enhancing (CASE) fusion protein comprising a target protein binding (TPB) domain and a transmembrane retention (TMR) domain, and optionally a chaperone machinery peptide (CMP) domain, wherein co-expression in a host cell in culture of the CASE fusion protein and a target protein enhances the level of target protein secreted from the host cell in the culture media compared to the target protein in the absence of the CASE fusion protein.
Target protein binding domain selection
The TPB domain is selected from receptor/ligand-binding portions, truncated receptors fused to Fc as trap molecules, PDZ domains such as NHERF/NHERF family members, and Fc-binding domains or peptides such as HSV-1 gE, Protein A, Protein G, and FcBP.
Transmembrane retention domain selection
The TMR domain is selected from transmembrane retention elements, including Types I-IV transmembrane proteins and named example transmembrane regions such as VSV-G and KDELR/KDEL receptor-derived regions, with domain orientation and linker arrangements described in the fusion architecture.
Chaperone machinery peptide domain
The CASE fusion protein optionally includes a CMP domain, including J-domain fragments or analogs and other chaperone machinery peptide elements with active-fragment constraints.
The claims cover a CASE fusion protein in which a TPB domain binds a target protein and a TMR domain retains the fusion in cells, with an optional CMP domain, such that target protein secretion into culture media is increased compared with target expression without the CASE fusion protein.
Stated Advantages
Enhances the level of target protein secreted from a host cell into culture media compared to expressing the target protein without the CASE fusion protein.
Avoids co-secretion of the CASE fusion protein into extracellular media while still enhancing target secretion.
An ER-retention dilysine (KK) motif in the TMR abolishes secretion enhancement.
Documented Applications
Co-expression in a host cell in culture to enhance secretion of a target protein, exemplified by IL13R-TF-Fc secretion quantified by Protein A/anti-IgG ELISA in HEK293 cells.
Enhancement of secretion for Fc-containing targets using CASE fusion protein configurations such as TPB(gE) with VSVG vs KDELR TMRs.
Increase of target secretion across multiple Fc drugs/targets.
Application to a human subject with AAT deficiency, where expression of the CASE fusion protein in the subject’s cells increases circulating AAT levels.
Restoration of deficient secretion in diseases, notably cystic fibrosis (CFTR).
Gene therapy and transgenic production contexts.
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