Site-specific modification of proteins through chemical modification enabling protein conjugates, protein dimer formation, and stapled peptides
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Publication Number
US-8927485-B2
Publication Date
2015-01-06
Expiration Date
Abstract
The present invention generally provides methods for the site-specific modification of peptides, polypeptides, and proteins, e.g., granulocyte macrophage colony-stimulating factor, human superoxide dismutase, annexin, leptin, antibodies and the like, cytokines and chemokines, at their N-termini and at sites at which unnatural aminoacids have been introduced along the protein framework. The modifications described herein can be used for the synthesis and application of the adducts in radio-labeling, molecular imaging and protein therapeutic applications, and the treatment of disorders such as rheumatoid arthritis, lupus erythematosus, psoriasis, multiple sclerosis, type-1 diabetes, Crohn's disease, and systemic sclerosis, Alzheimer disease, cancer, liver disease (e.g., alcoholic liver disease), and cachexia.
Core Innovation
The invention relates to site specifically producing a covalently linked multimeric protein. It provides proteins in the form of a multimer that are non-covalently bound to each other and enables covalent linkage using linker chemistry that targets defined sites at protein N-termini and/or unnatural amino-acid sites.
The disclosed framework combines proteins and linker chemistry so that coupling occurs at defined protein positions using carbonyl-generating substrates and carbonyl reactive functional groups. In one aspect, the method uses a bifunctional linker to form a covalent bond to a glycine or alanine residue at the N-terminus in each protein, and in another aspect it forms a covalent bond to an α-carbonyl amide moiety in each protein.
The described linkage strategy supports rapid formation of an α-carbonyl amide intermediate and enables subsequent irreversible coupling or proximity enhanced cycloaddition, including carbonyl/oxime-hydrazone/semicarbazone-type linkages for covalent attachment and multimerization/dimer formation. The partial content also describes experimental observations relevant to multimer formation, including PLP transamination of A-leptin to a ketoamide/pyruvyl protein and the resulting formation of mono-oxime and covalent dimers.
Claims Coverage
The independent claims cover two related methods for site specifically producing covalently linked multimeric proteins. The inventive features are: covalent N-terminal glycine or alanine linkage using a bifunctional carbonyl-containing linker, and covalent linkage via an α-carbonyl amide moiety using a bifunctional linker with two or more carbonyl reactive functional groups.
Covalent N-terminal multimer linkage via bifunctional carbonyl linker
providing one or more proteins comprising a four helix bundle protein or comprises a zipper binding motif, wherein the one or more proteins are non-covalently bound to each other within said multimer and comprise a polypeptide comprising a glycine or alanine residue at the N-terminus; combining a bifunctional linker and said one or more proteins, wherein said bifunctional linker forms a covalent bond to said glycine or alanine residue in each protein to produce a covalently linked multimer, and wherein said bifunctional linker comprises two or more carbonyl groups
Covalent linkage through an α-carbonyl amide moiety using carbonyl reactive bifunctional linker
providing one or more proteins in the form of a multimer, wherein said one or more proteins are non-covalently bound to each other within said multimer, each one of one or more proteins comprising an α-carbonyl amide moiety; combining a bifunctional linker and said one or more proteins, wherein said bifunctional linker (a) forms a covalent bond to the α-carbonyl amide moiety in each one of said one or more proteins and (b) comprises two or more carbonyl reactive functional groups
Across the independent claims, the coverage is directed to covalently linking non-covalently associated multimeric proteins using carbonyl-based bifunctional linkers that react at a defined N-terminal site (glycine or alanine) or at an α-carbonyl amide moiety to yield a covalently linked multimer.
Stated Advantages
Linker stability is described as improved for diketones compared with aldoximes, including stable adduct formation with bis-ketones.
The disclosed chemistry enables covalent coupling and ligation while using site-selective carbonyl chemistry to produce covalently linked multimeric proteins.
Documented Applications
Imaging agents with radiolabels are mentioned as biologically active agents that can be coupled using the described carbonyl chemistry.
Immunoconjugates are described as assembled using an antibody or antibody fragment in conjunction with the described linker and protein carbonyl condensation approach.
The partial content describes application to proteins such as cytokines and other biologically active agents, using carbonyl-based site-selective conjugation or ligation to generate multimeric protein products.
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