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-9675671-B2
Publication Date
2017-06-13
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 relates to engineered fusion proteins comprising an N-terminal leader formed by either SEQ ID NO:1 or residues 2-18 of SEQ ID NO:1 fused directly to the N-terminus of a cytokine. The described leader is used with cytokine variants, including an IGF-1 variant identified as “765IGF” (SEQ ID NO:2), and additional IGF-1 variant regions such as the R3-IGF region (SEQ ID NO:3 and variants).
The problem described is the need for improved cytokine-based targeting and performance for anti-cancer drug conjugates, including higher recombinant yield and better receptor binding or displacement compared with wild-type human IGF-1, together with improved drug loading and storage stability. The partial content reports that the 765IGF constructs show improved IGF receptor binding and displacement of wild-type IGF-1, while reducing soluble IGF-binding-protein affinity through changes in the IGF-1 variant region.
The invention further extends the N-terminal leader concept to other ErbB-1 ligands and cytokine fusion constructs, including a “765EGF” construct (SEQ ID NO:8) and a TNF-alpha fusion construct (“765TNFa”, SEQ ID NO:17). The partial content additionally describes covalent attachment chemistry concepts for chemotherapeutics to lysine, aspartate, and glutamate residues using carbodiimides such as EDC, forming amide bond linkages, and reports examples of drug-conjugates such as 765IGF-MTX and 765EGF-bendamustine with reported in vitro and in vivo activity.
Claims Coverage
The partial claim set includes two independent claims. Both claims are directed to fusion proteins defined by an N-terminal leader (SEQ ID NO:1 or residues 2-18 of SEQ ID NO:1) fused directly to the N-terminus of a cytokine, with claim-dependent coverage narrowing the cytokine selection to specific SEQ ID numbers or residue ranges and optionally adding receptor-ligand functional constraints.
N-terminal leader fused to cytokine N terminus
A fusion protein comprising either SEQ ID NO:1 or residues 2-18 of SEQ ID NO:1 fused directly to the N-terminus of a cytokine.
Leader fused to selected cytokine sequence or residue ranges
A fusion protein consisting of either SEQ ID NO:1 or residues 2-18 of SEQ ID NO:1 fused to the N terminus of a cytokine selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, residues 2-54 of SEQ ID NO:9, residues 40-89 of SEQ ID NO:10, residues 101-184 of SEQ ID NO:11, residues 63-148 of SEQ ID NO:12, or residues 32-111 of SEQ ID NO:13.
Across the independent claims, coverage centers on direct N-terminal fusion of SEQ ID NO:1 (or residues 2-18 thereof) to the N-terminus of a cytokine, with the cytokine identity defined by specified SEQ ID numbers or residue ranges. Dependent refinements further constrain cytokines to ligand functionality for ErbB-1 or IGFR1 and narrow specific fusion identities such as those involving SEQ ID NO:2 and defined residue segments.
Stated Advantages
Higher recombinant yield in microbial hosts.
Improved IGF receptor binding and displacement of wild-type IGF-1.
Higher methotrexate loading on 765IGF-MTX.
Improved storage stability versus other variants.
Documented Applications
In vitro inhibition of MCF7 proliferation by 765IGF-MTX.
In vivo activity of 765EGF-bendamustine in EGF receptor-positive xenograft models, including reported potency versus free bendamustine.
Reported curing in mice using 765EGF-bendamustine.
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