Peptide construct having a protease-cleavable linker
Inventors
Crowe, Scott • West, Mike • Roberts, Kevin
Assignees
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Publication Number
US-12234279-B2
Publication Date
2025-02-25
Expiration Date
Abstract
There is provided inter alia a construct suitable for oral administration comprising a first polypeptide and a second polypeptide connected by a labile peptide linker, wherein the labile peptide linker is labile to one or more proteases present in the intestinal tract and wherein the first and second polypeptides are substantially resistant to said one or more proteases.
Core Innovation
Oral-administered peptide/protein constructs are disclosed in which a first polypeptide and a second polypeptide are connected by a labile peptide linker. The polypeptides include immunoglobulin chain variable domains (ICVD) such as VHH and VH, and the labile peptide linker is designed to be labile to one or more proteases present in the intestinal tract while the polypeptides are substantially resistant to intestinal proteases.
The linker comprises a polypeptide sequence format with defined residue and segment parameters, including a format containing G and S repeating elements and a linker core containing B where B is K. The design uses labile sites that are protease-cleavable and distinguishes shielded labile sites versus non-shielded labile sites based on defined residue and segment rules, with cleavage functionality for protease activity in the intestinal tract including trypsin/trypsin-like and chymotrypsin/chymotrypsin-like proteases, and optionally MMP cleavage sites.
A set of lability and stability assessment assays are disclosed, including a Trypsin Protease Assay, a Chymotrypsin Protease Assay, and a Faecal Protease Assay. The disclosure defines desirable cleavage and uncleaved mass thresholds measured after mixing in the protease assays, and stability to yeast proteases is assessed in connection with producing the construct using a Yeast Expression Protocol in host yeast (S. cerevisiae).
Claims Coverage
The document contains two independent claims. Across them, the inventive coverage centers on a construct linking a first and second VHH using a protease-labile yet yeast-protease-stable labile peptide linker, together with specified linker sequence formats and parameter values tied to protease-lability and yeast stability performance thresholds.
Intestinally labile, yeast-protease-stable VHH/VHH construct with assay-defined lability and stability
A method of making a construct comprising a first VHH and a second VHH connected by a labile peptide linker that is labile to one or more proteases present in the intestinal tract, stable to yeast proteases, and wherein the construct comprises quantitative performance requirements including greater than 50% by mass of the construct cleaved into first and second VHHs after 160 minutes after mixing in the Trypsin Protease Assay and no more than 10% by mass of the construct cleaved into first and second VHHs after producing the construct using the Yeast Expression Protocol, and/or wherein the first and second VHHs are resistant to one or more proteases present in the intestinal tract such that at least 70% by mass of the first VHH and at least 70% by mass of the second VHH remain uncleaved after 10 minutes after mixing in the Trypsin Protease Assay.
Specified labile peptide linker format with B=K and defined parameters for VHH/VHH connection
The labile peptide linker comprises a polypeptide sequence of the format [-(G a S) x—B-(G b S) y—] z where a is 3 to 5, b is 3 to 5, x is 1 or 2, y is 1 or 2, z is 1 to 3, and B is K, and the method includes providing a host yeast cell capable of expressing the construct, producing the construct in the host yeast cell, and recovering the construct from the host yeast cell.
Fixed-parameter labile peptide linker format for intestinally labile, yeast-protease-stable VHH/VHH construct
A method of making a construct comprising a first VHH and a second VHH connected by a labile peptide linker wherein the labile peptide linker is labile to one or more proteases present in the intestinal tract, stable to yeast proteases, and the labile peptide linker comprises a polypeptide sequence of the format [-(G a S) x—B-(G b S) y—] z wherein a is 4, b is 4, x is 2, y is 2, z is 1, and B is K, and the method comprises providing a host yeast cell capable of expressing the construct, producing the construct in the host yeast cell, and recovering the construct from the host yeast cell.
Overall, the claim coverage requires a construct linking two VHHs with a labile peptide linker that is protease-labile in the intestinal tract while stable to yeast proteases. The inventive scope is further anchored by the specified linker polypeptide sequence formats with B=K and defined parameter values, and by assay-defined cleavage and uncleaved-mass performance requirements in the Trypsin Protease Assay and yeast production context.
Stated Advantages
Greater than 50% by mass of the construct is cleaved into first and second VHHs after 160 minutes after mixing in the Trypsin Protease Assay.
No more than 10% by mass of the construct is cleaved into first and second VHHs after producing the construct using the Yeast Expression Protocol.
At least 70% by mass of the first VHH and at least 70% by mass of the second VHH remain uncleaved after 10 minutes after mixing in the Trypsin Protease Assay.
Documented Applications
Therapeutic use for GI autoimmune/inflammatory disease, including Crohn's disease and ulcerative colitis.
Therapeutic use for C. difficile infection, including C. difficile toxin A and C. difficile toxin B.
Oral formulations, optionally enteric coated.
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