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Designed ankyrin repeat domains with improved stability

A repeat domain, ankyrin technology, applied in the field of disease treatment

Pending Publication Date: 2022-03-18
MOLECULAR PARTNERS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Despite the development of such engineered ankyrin repeat domains with binding specificity for serum albumin, there remains a need and a challenge to improve today's drug products, which generally have ankyrin repeat domains with binding specificity for serum albumin. protein repeat domain), and in particular storage stability properties

Method used

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  • Designed ankyrin repeat domains with improved stability
  • Designed ankyrin repeat domains with improved stability
  • Designed ankyrin repeat domains with improved stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Example 1: Construction of designer anchors with binding specificity for serum albumin and improved stability protein repeat domain

[0136]Unexpectedly, it was observed that a protein consisting of the amino acid sequence corresponding to SEQ ID NO: 2 (originally described in WO2016156596) appeared to be unstable after incubation at elevated temperatures (see Examples 2 to 6), despite the fact that it is a variant with improved stability compared to an earlier designed ankyrin repeat domain (WO2012 / 069654) with binding specificity for serum albumin. It is therefore an object of the present invention to provide variants of SEQ ID NO: 2 which exhibit improved stability after incubation at elevated temperatures while maintaining serum albumin binding and pharmacokinetic properties. Through a highly iterative process involving several rounds of changing amino acids at multiple positions (e.g., comparable to the alanine scanning process well known to those skilled in th...

Embodiment 2

[0138] Example 2: Expression and purification of proteins

[0139] Proteins consisting of SEQ ID NOs: 2 to 6, additionally having a His-tag SEQ ID NO: 1 fused to its N-terminus, and consisting of SEQ ID NOs: 7 to 12, additionally having a His-tag fused to its N-terminus were produced in E. coli The terminal His-tagged protein of SEQ ID NO: 1 was purified to homogeneity and stored in PBS buffer. For clarity, proteins #2 to #6 are designed ankyrin repeat domains with binding specificity for serum albumin and proteins #7 to #12 are designed anchor protein domains containing binding specificity for serum albumin Recombinant binding proteins of protein repeat domains. Proteins #7 and #8 contain twice as much SEQ ID NO:2. Proteins #9 and #10 contain twice as much SEQ ID NO:3. Proteins #11 and #12 contain twice as much SEQ ID NO:4. Protein #7 of SEQ ID NO: 134 from WO2016156596 is well known to those skilled in the art, and protein #8 of SEQ ID NO: 21 from WO2018054971 is well ...

Embodiment 3

[0141] Example 3: Storage Stability Incubation

[0142] The protein of Example 2 was tested for 1 week (7 days) storage stability at various pH at 100 micromolar protein concentration and 60°C. Buffers used were PBS (pH 7.4; 137 mM NaCl, 10 mM phosphate and 2.7 mM KCl) or phosphate / citrate (pH 5.7; 375 mM Na2HPO4*2H2O and 150 mM NaCl, pH adjusted using 1M citrate monohydrate ), or phosphate / citrate (pH 4.75; 30 mM citric acid and 30 mM NaH2PO4, pH adjusted with sodium hydroxide), or phosphate / citrate / borate (pH 8.5, 30 mM citric acid, 30 mM NaH2PO4 , 30mM boric acid). When the protein is mixed with the corresponding buffer, the resulting pH values ​​are pH 7.4, pH 6, pH 5 or pH 8.5, respectively. In parallel to the incubation at 60°C, an aliquot of the protein was incubated at -80°C for 1 week (7 days) as a control.

[0143] Stability during incubation at 60°C and at different pH is industrially relevant for the manufacture of designer ankyrin repeat domains or recombinan...

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Abstract

The present invention relates to designed ankyrin repeat domains with binding specificity for serum albumin, and in particular, to such designed ankyrin repeat domains with improved stability. The invention also relates to recombinant binding proteins comprising such designed ankyrin repeat domains, nucleic acids encoding such designed ankyrin repeat domains or proteins, pharmaceutical compositions comprising such proteins and the use of such proteins or pharmaceutical compositions in the treatment of disease.

Description

[0001] Cross references to related patent applications [0002] This application claims the benefit and priority of European Patent Application EP19178282 filed with the European Patent Office on 04.06.2019. The content of European Patent Application EP19178282 is hereby incorporated by reference in its entirety, including all tables, figures and claims. technical field [0003] The present invention relates to designer ankyrin repeat domains with binding specificity for serum albumin, and in particular, to such designer ankyrin repeat domains with improved stability. The present invention also relates to recombinant binding proteins comprising such designer ankyrin repeat domains, nucleic acids encoding such designer ankyrin repeat domains or proteins, pharmaceutical compositions comprising such proteins and such proteins or medicaments Use of the composition in the treatment of disease. Background technique [0004] For biopharmaceutical products where the active compone...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C07K19/00C12N15/62A61K38/16A61K48/00A61P35/00
CPCC07K14/001A61K48/005A61P35/00C07K2319/70C07K2319/21A61K38/00C07K14/47C07K14/435C07K14/765C07K2319/35
Inventor J·席林M·瓦尔泽
Owner MOLECULAR PARTNERS AG
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