Crosslinking of proteins and other entities via conjugates of α-haloacetophenones, benzyl halides, quinones, and their derivatives
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Publication Number
US-9770515-B2
Publication Date
2017-09-26
Expiration Date
Abstract
The present invention relates to the formation of conjugates (e.g., protein-protein dimers) using a-halo-acetophenones, benzylic halides, quinones, and related compounds as a conjugating system. The invention also features compositions that include the conjugates described herein, as well as uses of these conjugates in methods of medical treatment.
Core Innovation
The invention relates to conjugates formed by crosslinking proteins and biologically active agents using alpha-haloacetophenones, benzylic halides, and quinone-based electrophile systems. The conjugate includes an aryl or heteroaryl moiety A that links two entities through covalent and heteroatom-containing linkers, with optional substituted aryl and optionally substituted heteroaryl portions for ArZ1 and ArZ2.
The linker Z is a covalent bond, O, S, or NRZ1, and related linker components define optional polyethylene glycol segments and linking-group motifs. The conjugate further specifies that R is a globular protein and R' or R3 is a protein, a biologically active agent, or a biologically compatible agent, where each said protein comprises at least three amino acids linked by peptide bonds.
The disclosed chemistry also includes thioether-containing protein conjugation strategies, chemoselective oxime formation from carbonyls, reduction of alpha-carbonyl groups to hydroxy groups, and formation of beta-thio-acryloyl adducts by alkylation of free cysteines with acrylic acid derivatives. Documented embodiments relate to site-specific protein conjugates built on electrophilic quinone, alpha-haloacetyl, and related aromatic cores, with covalently tethered multiple proteins via site-specific linkers.
Claims Coverage
The consolidated claim coverage centers on one independent claim defining a highly specified conjugate architecture that links an optionally substituted aryl/heteroaryl scaffold to a globular protein R and to an additional protein or biologically active or biologically compatible agent R' or R3 through a defined variable linker with optional polyethylene glycol and defined connectivity elements.
Specified conjugate with aryl/heteroaryl scaffold linked to globular protein and agent
A conjugate having a structure in which A is optionally substituted aryl, optionally substituted heteroaryl, or a substructure according to an ArZ1-Z-ArZ2 framework, with Z defined as a covalent bond, O, S, or NRZ1; the linker includes defined options for XZ1, QZ1, L1, L2, Q1, Q2, and optional polyethylene glycol segments; and wherein R is a globular protein and R' or R3 is a protein, a biologically active agent, or a biologically compatible agent.
Globular protein comprising at least three peptide-bonded amino acids
Each said protein independently comprises at least three amino acids linked by peptide bonds, with R being a globular protein as part of the conjugate structure.
The claim coverage centers on a highly specified conjugate structure that fixes the relationship between an optionally substituted aryl/heteroaryl scaffold and a globular protein R plus an additional protein or biologically active/biologically compatible agent R' or R3 using a defined variable linker with optional polyethylene glycol and defined connectivity elements.
Stated Advantages
Enables delivering a therapeutic agent to cells undergoing necrosis or apoptosis.
Provides a pharmaceutical composition including the conjugate with a pharmaceutically acceptable excipient.
Improved stability of reduced benzylic alcohol conjugates.
Provides conjugates and methods for modifying proteins with selected payloads.
Supports attachment of therapeutic, diagnostic, imaging, and biologically compatible agents.
In the documented annexin:IL-10 studies, the conjugates show increased duration of action and less systemic toxicity.
The reported in vivo results include attenuation of synovial volume, improved lymph flow, reduced inflammation, and reduced bone and cartilage damage.
Documented Applications
Delivering a therapeutic agent to cells undergoing necrosis or apoptosis by contacting cells or tissue with the conjugate agent.
Use of the conjugate in a pharmaceutical composition with a pharmaceutically acceptable excipient.
Protein conjugation of globular proteins with additional proteins, biologically active agents, or biologically compatible agents.
Modification of proteins containing free cysteine residues or proteins modified to include free cysteine residues.
Use with annexin proteins, antibodies, cytokines, polymers, nucleic acids, carbohydrates, small molecule therapeutic agents, imaging agents, and diagnostic agents.
In vivo evaluation of annexin:IL-10 conjugates in TNF-transgenic arthritis mice using MRI, lymphatic imaging, histologic analysis, and immunohistochemistry.
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