Systems, compositions, and methods for transplantation
Inventors
HANNA, Lewis • Laughlin, John David • BROWNING, Shawn Robert
Assignees
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Publication Number
US-10940189-B2
Publication Date
2021-03-09
Expiration Date
Abstract
Systems and methods for purification and concentration of autologous alpha-2-macroglobulin (A2M) from whole blood are provided. Also provided are diagnostic methods for identifying sites in the synovial joints, spine, tendons or ligaments for treatment of pain, degeneration, or inflammation with autologous A2M. Methods for utilizing autologous A2M in combination with other autologous treatments (e.g. platelets and other growth factors) are provided in addition to combinations with exogenous drugs or carriers. Also provided is a method of producing recombinant A2M wild type or variants thereof where the bait region was modified to enhance the inhibition characteristics of A2M and/or to prolong the half life of the protein in joints and spine disc or epidural space.
Core Innovation
The invention relates to systems and methods to concentrate alpha-2-macroglobulin (A2M) from patient blood. The approaches include filtration selected from dead-end filtration and tangential flow filtration, with optional preprocessing by centrifugation of patient blood to obtain supernatant and then concentrating A2M from the supernatant.
The invention provides a framework for treating spine/joint/inflammation-related pain using autologous alpha-2-macroglobulin (A2M), including liquid autologous A2M compositions enriched at least 1.1× versus whole blood with reduced low-molecular-weight proteins. The described approaches use filtration/ultrafiltration and systems such as hollow-fiber or tangential flow filtration to remove cells and retain or increase A2M while lowering <500 kDa components.
The invention further describes recombinant variant A2M constructs with modified bait regions, including multiple protease recognition sites and non-natural sites, to enhance protease inhibition and/or prolong half-life. The document also discusses A2M biology and mechanisms for pain and inflammation, including a bait-region trapping mechanism and protease inhibition, together with concepts associated with inhibition of FAC formation.
Claims Coverage
The provided independent claim set includes 1 independent claim, and the claim coverage centers on a recombinant alpha-2-macroglobulin (A2M) polypeptide with a non-natural bait region. The inventive features include replacement of the wild-type bait region, sequence identity and contiguity requirements relative to SEQ ID NO: 47, and two or more protease recognition sequences; dependent claims further refine identity thresholds and add optional glycosylation, PEG, and covalent protease attachment capabilities.
Recombinant A2M with a non-natural bait region replacing wild-type bait region
A recombinant alpha-2-macroglobulin (A2M) polypeptide comprising a non-natural bait region that replaces the bait region of a wild-type A2M polypeptide with a sequence according to SEQ ID NO: 3.
SEQ ID NO: 47 identity and contiguous match
The non-natural bait region comprises a sequence with 100% sequence identity to at least 24 contiguous amino acid residues of SEQ ID NO: 47 and has at least 70% identity to the entirety of SEQ ID NO: 47.
Two or more protease recognition sequences
The non-natural bait region comprises two or more protease recognition sequences.
Overall, the claim coverage centers on a recombinant A2M polypeptide whose bait region is replaced by a non-natural bait region meeting specified identity and contiguity requirements relative to SEQ ID NO: 47 and containing two or more protease recognition sequences, with dependent refinements tightening identity thresholds and optionally adding glycosylation and PEG at an added glycosylation site and covalent protease attachment.
Stated Advantages
Liquid autologous A2M compositions are enriched at least 1.1× versus whole blood with reduced low-molecular-weight proteins.
The approaches remove cells and retain or increase A2M while lowering <500 kDa components.
Recombinant variant A2M constructs are described as enhancing protease inhibition and/or prolonging half-life.
Documented Applications
Concentrating A2M from patient blood using filtration, including dead-end filtration and tangential flow filtration, optionally after centrifugation to provide supernatant.
Treating spine/joint/inflammation-related pain using autologous alpha-2-macroglobulin (A2M).
Pain and inflammation contexts associated with A2M biology, including bait-region trapping and protease inhibition mechanisms.
Therapeutic, diagnostic, and assay contexts for A2M-containing compositions.
Concepts relating to inhibition of FAC formation in the broader A2M context described in the document.
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