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Peptide construct having a protease-cleavable linker

Inactive Publication Date: 2018-02-08
VHSQUARED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about creating new types of proteins that can be taken as a pill and broken down in the stomach and intestinal tract. These proteins can be used to treat diseases such as inflammatory bowel disease and infection from harmful microbes. The proteins are connected by a labile peptide linker that is sensitive to proteases in the stomach and intestine. This helps to release the individual components of the protein in specific regions of the digestive system. This invention provides a convenient way to design oral medications that can be tailored to target specific parts of the gastrointestinal tract.

Problems solved by technology

Direct fusion of polypeptides without a linker may lead to many undesirable outcomes including misfolding of the fused polypeptides, low yield in polypeptide production or impaired bioactivity.
However, stable linkers also have several potential drawbacks including steric hindrance between polypeptides, decreased bioactivity, and altered biodistribution (Chen et al.

Method used

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  • Peptide construct having a protease-cleavable linker
  • Peptide construct having a protease-cleavable linker
  • Peptide construct having a protease-cleavable linker

Examples

Experimental program
Comparison scheme
Effect test

example 1

in Protease Assay

[0534]To test the lability of labile peptide linkers, the Trypsin Protease Assay was developed. This assay is performed as follows.

[0535]A buffered (10 mM acetic acid, pH 3.2, containing 0.01% thimerosal) aqueous suspension of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated Trypsin-agarose beads (trypsin from bovine pancreas; T4019; Sigma Aldrich) is used for the assay. The beads are washed 3 times with water (250 μl beads+1.25 ml water) followed by washing 5 times with Trypsin buffer (TRYP buffer; 1 mM Tris-HCl, 20 mM CaCl2 [pH 8.0]). Finally, the resin is resuspended in TRYP buffer as a 50% (v / v) suspension.

[0536]100 μl of a 2 mg / ml construct solution is mixed with 225 μl 50% (v / v) immobilized TPCK-treated Trypsin in TRYP buffer. After time intervals such as 0, 10, 15, 30, 45 and 60 minutes of incubation at 37° C. in a shaker, samples are taken as follows: resin is pelleted by a 1 min centrifugation step at 500×g, and a 40 μl sample is taken from t...

example 2

trypsin Protease Assay

[0538]The Trypsin Protease Assay protocol can be used wherein Trypsin beads are substituted with Chymotrypsin beads.

example 3

l Extract Protease Assay

[0539]To test the lability of labile peptide linkers, the Faecal Extract Protease Assay was developed. Faecal extract is a physiologically relevant matrix in particular for polypeptides targeted to the large intestine. This assay is performed as follows.

[0540]Human faecal samples from multiple individuals are turned into slurries with addition of 1×PBS at a ratio of 1, 2 or 3 mLs 1×PBS per gram of faeces. The slurries are then pooled (such that one pool represents the combined protease output from the faeces of multiple individuals), centrifuged and the supernatants removed, aliquoted and stored at −70 degrees C. This process removes the faecal matrix, including any cellular material. For digestion, constructs are incubated at a concentration of 160.16 μg / ml at 37° C. in pooled human faecal supernatant for 60 mins in the absence of BSA carrier. Aliquots are taken after time intervals such as 0, 10, 30 and 60 minutes and are mixed 1:1 in Protease Stop Solution...

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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.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present application is a continuation application of PCT / EP2016 / 057022 filed Mar. 31, 2016 which claims priority from EP 15162114.1 filed Mar. 31, 2015, EP 15162115.8 filed Mar. 31, 2015, EP 15162112.5 filed on Mar. 31, 2015 and EP 16152320.4 filed on Jan. 21, 2016, the contents of each of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION[0002]The present invention relates to constructs suitable for oral administration comprising polypeptides connected by a labile peptide linker as well as to pharmaceutical compositions comprising such constructs. The present invention also relates to methods for preparing such constructs, methods which assay the lability of such constructs, methods which utilise such constructs, nucleic acids encoding such constructs, cDNA and vectors comprising nucleic acids encoding such constructs, host cells expressing or capable of expressing such constructs and to uses of such ...

Claims

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Application Information

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IPC IPC(8): C07K16/12C07K16/24
CPCC07K16/1282C07K16/241C07K2319/50C07K2317/56C07K2317/94A61K39/395C07K16/2878C07K16/468C07K2319/00C07K2317/64A61P1/00A61P1/04A61P29/00A61P31/04A61P37/00A61P37/06C07K16/24
Inventor CROWE, SCOTTWEST, MIKEROBERTS, KEVIN
Owner VHSQUARED
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