Human tissue kallikrein 1 glycosylation isoforms
Inventors
Charles, Matthew • Kolodka, Tadeusz • Williams, Mark
Assignees
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Publication Number
US-9364521-B2
Publication Date
2016-06-14
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 a composition comprising a first mature human tissue kallikrein-1 (KLK1) polypeptide and a second mature human tissue kallikrein-1 (KLK1) polypeptide with different N-glycosylation patterns. The first mature human KLK1 polypeptide has three Asn-linked glycans attached at residues 78, 84, and 141 as defined by SEQ ID NO:1, and the second mature human KLK1 polypeptide has two Asn-linked glycans attached at residues 78 and 84 but not 141 as defined by SEQ ID NO:1.
The two mature human KLK1 polypeptides are present in the composition in a ratio ranging from 45:55 to 55:45. The composition is substantially free of other glycosylated isoforms of KLK1 and includes characterization concepts such as purity and aggregation thresholds, SEC HPLC single-peak criteria, endotoxin, host cell protein (HCP), and host cell DNA.
The document provides functional evidence that the specified KLK1 glycoform mixture stimulates glucose uptake from the circulation and/or by tissues with increased activity compared to a composition having a different mixture ratio. Functional data described include hyperinsulinemic-euglycemic clamp results in Sprague-Dawley rats, where a rhKLK1 glycoform mixture at approximately a 50:50 ratio yields a higher glucose infusion rate AUC than either isolated double or triple glycoforms and than human urinary KLK1 with a 60:40 ratio, as well as glucose lowering in a db/db mouse type 2 diabetes model with the specified glycoform ratio.
Claims Coverage
The partial content includes two independent claims. Across the independent claims, the inventive features include two mature human KLK1 polypeptides with different N-glycosylation patterns, the defined glycan attachment positions at residues 78, 84, and 141 versus residues 78 and 84 only, a 45:55 to 55:45 ratio, and substantially free of other glycosylated isoforms of KLK1 with increased glucose uptake activity versus an about 60:40 composition.
Two mature KLK1 polypeptides with distinct glycosylation patterns
The first mature human KLK1 polypeptide has three Asn-linked glycans at residues 78, 84, and 141 as defined by SEQ ID NO:1, and 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.
Controlled 45:55 to 55:45 mixture ratio
The first mature human KLK1 polypeptide and the second mature human KLK1 polypeptide are present in the composition in a ratio ranging from 45:55 to 55:45.
Residue range inclusion (25-262) for SEQ ID NO:1 or SEQ ID NO:2
The mature human KLK1 polypeptides comprise residues 25-262 of SEQ ID NO:1 or SEQ ID NO:2.
Substantially free of other glycosylated KLK1 isoforms and increased glucose uptake activity versus 60:40
The composition is substantially free of other glycosylated isoforms of KLK1 and demonstrates an increased activity to stimulate glucose uptake from the circulation and/or by tissues in comparison to a composition wherein the first mature human KLK1 polypeptide and the second mature human KLK1 polypeptide are present in a ratio of about 60:40.
Overall, the independent claims are directed to a KLK1 composition that combines two mature human KLK1 polypeptides with specific Asn-linked glycans at residues 78 and 84, and optionally 141, and sets the mixture ratio to 45:55 to 55:45. The second independent claim further requires substantially no other glycosylated isoforms and ties the defined mixture to increased glucose uptake activity versus an about 60:40 composition.
Stated Advantages
Increased activity to stimulate glucose uptake from the circulation and/or by tissues compared to a composition with a ratio of about 60:40.
Documented Applications
A composition and/or device suitable for subcutaneous delivery of the KLK1 glycoform mixture.
Hyperinsulinemic-euglycemic clamp functional testing in Sprague-Dawley rats, assessing glucose infusion rate AUC.
A db/db mouse type 2 diabetes model, with reported glucose lowering and comparison to defined glycoform conditions.
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