Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Polypeptides

Inactive Publication Date: 2018-01-11
VHSQUARED
View PDF6 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides improves polypeptides made up of immunoglobulin chain variable domains that can be taken as a pill for diseases of the gastrointestinal tract, like inflammatory bowel disease. These improvements are made by substituting certain amino acids with histidine residues, which increases their stability in the intestines. These improvements make the polypeptides more effective and easier to develop as a drug for the treatment of gastrointestinal diseases.

Problems solved by technology

Many therapeutic polypeptides are, however, unstable in the intestinal tract and therefore the beneficial effect obtained from oral administration is generally limited (Bruno et al 2013 Ther Deliv4(11):1443-1467).
The effectiveness of these additional cysteine bridges on increasing proteolytic stability was highly dependent on the specific protease concerned and in some circumstances these additional cysteine bridges were detrimental to recombinant production levels.
However, in the context of immunoglobulin chain variable domains, single substitutions in amino acid sequence may detrimentally impact binding capability.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Polypeptides
  • Polypeptides
  • Polypeptides

Examples

Experimental program
Comparison scheme
Effect test

example 1

Standard Intestinal Tract Models, Standard Intestinal Stability Assays and Standard Potency Assays

[0258]The intestinal stability and potency of a polypeptide comprising an immunoglobulin chain variable domain can be assayed using the following methods. The methods below refer to

[0259]ICVDs, but are equally applicable to any polypeptide which comprises an ICVD, such as an antibody.

[0260]1.1 Standard Intestinal Tract Models

[0261]Ex vivo samples from human faeces and mouse small intestine samples are highly relevant matrices for estimation of stability in the human intestinal tract. Such samples contain native host-produced, and associated microbial-produced, proteases along with any chaotropic agents or surfactants that may influence ICVD stability in the presence of proteases. The enzymatic cleavage sites of at least some proteases present in the small intestine from murine and human origin are well characterised and conserved between the two species. Murine small intestinal supernat...

example 2

Substitution of a Lysine Residue with Alanine, Histidine or Glutamine in CDR2 of an Anti-TNF-alpha ICVD

[0525]Q65B1 is an anti-TNF-alpha ICVD isolated, cloned and purified from a llama immunised with soluble human recombinant TNF-alpha. Residue K59 of the Q65B1 polypeptide sequence was substituted with alanine, histidine or glutamine and the impact of each substitution on intestinal tract stability and potency was tested.

[0526]DNA encoding each ICVD was cloned into vector pMEK222, expressed, and purified from the periplasm of E. coli (either by Talon or Nickel NTA column). All ICVDs tested here carry an identical C-terminal Flag-His6 tag.

[0527]Residue K59 resides in CDR2 of Q65B1. Q65B1 with a K59A substitution is labelled “ID43F”, Q65B1 with a K59H substitution is labelled “ID8F-EV”, and Q65B1 with a K59Q substitution is labelled “ID44F”.

[0528]2.1.1 Potency—Standard TNFR2 / TNF Interference ELISA Assay—Experiment 1

[0529]Dose-response curves of each ICVD were generated using the Standa...

example 3

Substitution of a Lysine Residue with a Histidine Residue in both CDR2 and CDR3 of an anti-TNF-alpha ICVD

[0543]Both residues K59 and K101 of Q65B1 were substituted with histidine (making “ID34F”). Residue K59 resides in CDR2 of Q65B1 and residue K101 resides in CDR3 of Q65B1. DNA encoding ID34F was cloned and expressed in yeast.

[0544]Q65B1 substituted with a K59H residue (as in Example 2) was produced again, having the same sequence as ID8F-EV described above. However, on this occasion DNA encoding this ICVD was cloned and expressed in yeast (therefore lacking the C-terminal Flag-His6 tag) and is therefore labelled “ID32F” in this example.

[0545]3.1 Potency—Standard TNFR2 / TNF Interference ELISA Assay

[0546]Dose-response curves of each ICVD were generated using the Standard TNFR2 / TNF Interference ELISA Assay. A concentration range of 0-3 nM was used (FIG. 4).

[0547]3.2.1 Intestinal stability—Standard Mouse Small Intestinal Supernatant Intestinal Tract Model

[0548]ICVDs were digested for ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Fractionaaaaaaaaaa
Fractionaaaaaaaaaa
Fractionaaaaaaaaaa
Login to View More

Abstract

There is provided inter alia a polypeptide comprising an immunoglobulin chain variable domain comprising three complementarity determining regions (CDR1-CDR3) and four framework regions, wherein: (a) at least one lysine residue in CDR1, CDR2 and / or CDR3 has been substituted with at least one histidine residue, and / or (b) at least one arginine residue in CDR1, CDR2 and / or CDR3 has been substituted with at least one histidine residue; wherein the polypeptide has increased intestinal stability relative to a corresponding polypeptide not having said histidine substitutions.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present application is a continuation application of PCT / EP2016 / 057024 filed Mar. 31, 2016 which claims priority from EP 15162115.8 filed Mar. 31, 2015 and EP 16152320.4 filed 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 polypeptides comprising a region which is capable of binding a target with high affinity, especially those comprising immunoglobulin chain variable domains (ICVD) as well as to constructs comprising said polypeptides and pharmaceutical compositions comprising such polypeptides and constructs. The polypeptides, constructs and pharmaceutical compositions of the invention are all suitable for oral administration. The present invention also relates to methods of increasing the intestinal stability of a polypeptide comprising an immunoglobulin chain variable domain, methods of making a polypeptide comprising ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K16/12C07K16/24C07K16/28
CPCC07K16/1282C07K16/241C07K16/2866C07K2317/565C07K2317/567C07K2317/94C07K2317/622C07K2317/55C07K2317/54C07K2317/569C07K2317/92C07K2317/35A61K2039/542C07K2317/22A61P1/00A61P1/04A61P29/00A61P31/00A61P31/04A61P37/00A61P37/06Y02A50/30C07K16/12
Inventor CROWE, SCOTTWEST, MIKEROBERTS, KEVINCARLTON, TIM
Owner VHSQUARED
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com