Human tissue kallikrein 1 glycosylation isoforms
Inventors
Charles, Matthew • Kolodka, Tadeusz • Williams, Mark
Assignees
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Publication Number
US-9839678-B2
Publication Date
2017-12-12
Expiration Date
Abstract
Provided are preparations of tissue kallikrein-1 (KLK1) glycoforms having a defined number of oligosaccharide units per KLK1 molecule. Also provided are mixtures of such glycoforms, pharmaceutical compositions containing such glycoforms or mixtures thereof, methods of obtaining the rhKLK1 glycoforms, and therapeutic uses thereof.
Core Innovation
The invention relates to KLK1 glycosylation isoforms defined by the number and positions of N-linked glycans on mature human tissue kallikrein-1 (KLK1) polypeptides. A first mature human KLK1 polypeptide has three Asn-linked glycans at residues 78, 84, and 141 as defined by SEQ ID NO:1, and a second mature human KLK1 polypeptide has two Asn-linked glycans at residues 78 and 84 but not 141 as defined by SEQ ID NO:1.
A composition is provided that comprises both the first and the second mature human KLK1 polypeptides, with the two polypeptides present in a ratio ranging from about 45:55 to about 55:45. The disclosed isoform mixture is presented as a defined glycoform composition rather than a single glycosylated isoform, and the composition can be substantially free of other glycosylated isoforms of KLK1.
The invention also addresses compositions characterized by low endotoxin and host cell protein and/or host cell DNA, and reduction of aggregates as supported by SEC-HPLC single peak purity. In vivo evidence is described using an hyperinsulinemic-euglycemic clamp in rats, where unexpectedly higher glucose infusion rate AUC is reported for a 50:50 rhKLK1 mixture compared to single glycoforms and compared with human urinary KLK1.
Claims Coverage
Independent claim clm-00001 covers a method of treating an ischemic condition by administering a defined composition containing two mature human KLK1 polypeptides with different glycosylation patterns and present in a ratio from about 45:55 to about 55:45. The claim structure specifies the glycan attachment sites and limits the mixture to a dual-isoform composition defined by SEQ ID NO:1 features.
Dual glycoform mature human KLK1 composition for treatment
A method of treating an ischemic condition in a subject in need thereof by administering a composition comprising a first mature human KLK1 polypeptide with three glycans at three different positions and a second mature human KLK1 polypeptide with two glycans at two different positions.
Three-glycan KLK1 polypeptide defined by residues 78, 84, and 141
The first mature human KLK1 polypeptide has three Asn-linked glycans at residues 78, 84, and 141 as defined by SEQ ID NO:1.
Two-glycan KLK1 polypeptide defined by residues 78 and 84 but not 141
The second mature human KLK1 polypeptide has two Asn-linked glycans at residues 78 and 84 but not 141 as defined by SEQ ID NO:1.
Defined 45:55 to 55:45 KLK1 ratio
The first mature human KLK1 polypeptide and the second mature human KLK1 polypeptide are present in the composition in a ratio ranging from about 45:55 to about 55:45.
Across the disclosed claim coverage, the inventive focus is on treating ischemic conditions using a composition that combines two mature human KLK1 glycoforms defined by specific Asn-linked glycan attachment sites, including three-glycan at residues 78/84/141 and two-glycan at residues 78/84 but not 141, in a defined mixture ratio of about 45:55 to about 55:45.
Stated Advantages
Unexpectedly higher glucose infusion rate AUC for a 50:50 rhKLK1 mixture versus single glycoforms and versus human urinary KLK1.
Dose-dependent reduction in fasting blood glucose in db/db mice for a specific rhKLK1 glycoform mixture.
Documented Applications
Treating an ischemic condition in a subject in need thereof, including brain, cardiac, ischemic colitis, limb, or cutaneous ischemia.
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