Therapeutic variant alpha-2-macroglobulin compositions
Inventors
HANNA, Lewis • Laughlin, John David • BROWNING, Shawn Robert
Assignees
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Publication Number
US-10400028-B2
Publication Date
2019-09-03
Expiration Date
Abstract
A2M polypeptide compositions containing a non-natural bait region are disclosed. Methods of producing wild-type and variant A2M polypeptides and polynucleotides containing a non-natural bait region are also disclosed. The bait regions of the variant A2M polypeptides demonstrate enhanced protease inhibitory characteristics compared to wild-type A2M. Variant A2M polypeptides that demonstrate longer half-lives upon administration to an organism compared to wild-type A2M are disclosed. The A2M compositions are useful in treating a number of diseases and conditions including inflammation, chronic wounds, and diseases with a pathology associated with proteases.
Core Innovation
The invention relates to therapeutic alpha-2-macroglobulin (A2M) variants in which A2M is engineered with a non-natural bait region containing protease recognition sites. The non-natural bait region is defined by sequence identity to a specified SEQ ID NO and is used to target proteases for inhibition by the recombinant A2M polypeptide.
The disclosed non-natural bait regions can further include a suicide inhibitor element for covalent protease attachment. This protease-targeting design is presented in the context of inhibiting protease activity associated with extracellular matrix degradation and fibronectin-aggrecan complex formation, including effects on cartilage degradation.
Variant A2M compositions are described as recombinant polypeptides or encoding polynucleotides, including configurations characterized by enhanced protease inhibitory effectiveness versus wild-type A2M. The disclosure also includes variant-related features associated with longer in vivo half-life, including altered or abnormal glycosylation sites functionalized with PEG.
Claims Coverage
The independent claim is directed to a composition containing a recombinant A2M polypeptide with a non-natural bait region defined by sequence identity to a specified SEQ ID NO, with at least 80% identity. The claim coverage also includes suicide inhibitor-mediated covalent protease attachment, higher identity thresholds, consensus sequences for selected proteases, enhanced protease inhibition versus wild-type, and an encoding recombinant A2M polynucleotide.
Recombinant A2M with non-natural bait region identity
A composition comprising a recombinant alpha-2-macroglobulin (A2M) polypeptide comprising a non-natural bait region, wherein the non-natural bait region comprises a sequence with at least 80% identity to SEQ ID NO: 20.
Suicide inhibitor covalently attaching protease
A non-natural bait region includes a suicide inhibitor that covalently attaches a protease to a recombinant A2M polypeptide.
Bait region consensus for selected protease
The non-natural bait region includes a consensus sequence for a selected protease chosen from a specified group of proteases, including matrix metalloproteinase (MMP), ADAMTS, chymotrypsin, trypsin, elastase, compliment factors, clotting factors, thrombin, plasmin, subtilisin, Neprilysin, Procollagen peptidase, Thermolysin, Pregnancy-associated plasma protein A, Bone morphogenetic protein 1, Lysostaphin, Insulin degrading enzyme, ZMPSTE2, ZMPSTE4, ZMPSTE24, and acetylcholinesterase.
Higher identity threshold to SEQ ID NO: 20
The composition includes a non-natural bait region defined by a sequence having at least 90% identity to SEQ ID NO: 20.
Enhanced protease inhibition versus wild-type
A recombinant A2M polypeptide provides enhanced protease inhibition compared with wild-type A2M, where the inhibited protease is one or more specified classes selected from serine protease, threonine protease, cysteine protease, aspartate protease, metalloprotease, and glutamic acid protease, and combinations thereof.
Encoding recombinant A2M polynucleotide
The composition comprises a recombinant A2M polynucleotide encoding a recombinant A2M polypeptide as defined by the recombinant A2M composition with the non-natural bait region identity requirement.
Overall, the claim coverage focuses on recombinant A2M compositions with a non-natural bait region defined by sequence identity to SEQ ID NO: 20, optionally strengthened by higher identity thresholds, suicide inhibitor-mediated covalent protease attachment, and consensus bait sequences selected for specified protease sets. Additional coverage asserts enhanced protease inhibition compared with wild-type A2M and extends the composition to encoding recombinant A2M polynucleotides.
Stated Advantages
Enhanced protease inhibitory effectiveness compared with wild-type A2M.
Increased ability to prevent fibronectin-aggrecan complex (FAC) formation.
Longer in vivo half-life, including via abnormal glycosylation sites functionalized with PEG.
Documented Applications
Therapeutic use in inflammation and spine/joint pain.
Therapeutic use for extracellular matrix degradation, including cartilage/disc degeneration and associated FAC-related cartilage degradation context.
Therapeutic use for chronic wounds.
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