Fusion proteins containing insulin-like growth factor-1 and epidermal growth factor and variants thereof and uses thereof
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Publication Number
US-10391147-B2
Publication Date
2019-08-27
Expiration Date
Abstract
Fusion proteins comprising cytokines, particularly insulin-like growth factor-1 (IGF-1) and variants thereof, epidermal growth factor (EGF), and other ligands to the EGF receptor, are provided. The fusion proteins further comprise SEQ ID NO:1 or other segments having lysine, glutamic acid, or aspartic acid residues. Uses for the fusion proteins are also provided.
Core Innovation
The invention describes fusion or constructs in which an IGF-1R ligand is fused to another protein. The IGF-1R ligand is described using an IGF-1 variant termed 765IGF that includes a leader sequence from SEQ ID NO:1 and an R3-IGF segment from SEQ ID NO:2. The construct is presented as a fusion polypeptide in which SEQ ID NO:1 or residues 2-18 of SEQ ID NO:1 is fused directly to the N-terminus of a protein.
The invention also describes fusion constructs using EGF/ErbB-1 ligands, in particular a construct termed 765EGF that includes the leader sequence of SEQ ID NO:1 fused to a mature soluble EGF precursor segment. The constructs are described in relation to ligand binding to IGF-1R and ErbB-1/EGFR, including retaining IGF-1R affinity while reducing binding to soluble IGF binding proteins. Variants are also described using sequence identities and specified SEQ ID segments.
In addition to polypeptide fusion, the document describes compounds in which an anti-cancer chemotherapeutic agent is covalently attached to the fusion construct, including 765IGF-MTX and 765EGF-bendamustine. The document describes coupling through reactive groups and covalent attachment preferably involving lysine side chains and amide bonds. The constructs are reported to be refolded into active form and to exhibit improved storage stability, improved binding characteristics with reduced soluble IGF binding protein binding, and reported in vitro and in vivo anti-cancer outcomes including tumor growth inhibition and xenograft tumor cures.
Claims Coverage
The independent claim is clm-00001. It covers fusion polypeptides with an N-terminal IGF-1R ligand segment derived from SEQ ID NO:1 or residues 2-18 of SEQ ID NO:1 fused directly to a protein. The claim set includes one independent claim and dependent refinements that constrain the identity and ligand-binding nature of the fused protein.
N-terminal fusion of SEQ ID NO:1 IGF-1R ligand to an additional protein
A fusion polypeptide in which SEQ ID NO:1 or residues 2-18 of SEQ ID NO:1 is fused directly to the N-terminus of a protein.
High-identity variant with IGF-1R or ErbB-1 ligand function
The fusion polypeptide wherein the fused protein is a variant that is at least 90% identical to specified sequences and includes a ligand that binds IGF-1R or ErbB-1.
Specific residue-range constraint for the fused protein variant
The fusion polypeptide wherein the fused protein variant is at least 90% identical to specified residue ranges from SEQ ID NOs: 9, 10, 11, 12, or 13.
Overall, the claims cover fusion polypeptides defined by an N-terminal SEQ ID NO:1-derived segment directly fused to an additional protein, with dependent coverage limited to variants defined by at least 90% identity, specified residue regions, and ligand binding to IGF-1R or ErbB-1.
Stated Advantages
Improved microbial expression and purification yield.
Refolding into an active form.
Reduced binding to soluble IGF binding proteins while retaining IGF-1R affinity.
Higher chemotherapeutic loading.
Increased storage stability.
In vitro and in vivo efficacy, including inhibition of tumor cell proliferation and xenograft tumor cures in mice.
Documented Applications
Cancer treatment and inhibition, including treating cancer using constructs such as 765IGF-MTX and 765EGF-bendamustine, and timing with radiation or chemotherapy as described.
Use of the constructs to evaluate IGF-1R binding competition, including MCF7 binding competition.
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