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Novel ankyrin repeat binding proteins and their uses

a technology of ankyrin repeat and binding protein, which is applied in the field of recombinant binding proteins, can solve the problems of increasing the difficulty of treatment options

Pending Publication Date: 2022-07-28
MOLECULAR PARTNERS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention relates to a recombinant binding protein that includes an ankyrin repeat domain. These domains are found in various proteins and have a specific structure. The invention provides several embodiments, each with different ankyrin repeat domains that have a high degree of sequence identity with each other. The recombinant binding protein can be used for various applications, such as in research and development, and can be produced using routine experimentation. The technical effect of the invention is the provision of a versatile recombinant binding protein that can be used for various applications.

Problems solved by technology

Once the cell is infected with the coronavirus, treatment options become more difficult as the immune system (or therapeutic agent) must only target virus-infected cells, without damaging non-infected cells.

Method used

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  • Novel ankyrin repeat binding proteins and their uses
  • Novel ankyrin repeat binding proteins and their uses
  • Novel ankyrin repeat binding proteins and their uses

Examples

Experimental program
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Effect test

example 1

of Binding Proteins Comprising an Ankyrin Repeat Domain with Binding Specificity for SARS-CoV-2 Spike Protein

[0387]Summary

[0388]Using ribosome display (Hanes, J. and Plückthun, A., PNAS 94, 4937-42, 1997), multiple ankyrin repeat domains with binding specificity for different domains of the SARS-CoV-2 spike protein (RBD domain; S1 NTD domain; S2 domain) were selected from DARPin® libraries in a way similar to the one described by Binz et al. 2004 (loc. cit.), with specific conditions and additional de-selection steps. The binding and specificity of the selected clones towards recombinant SARS-CoV-2 spike protein target domains were assessed by E. coli crude extract Homogeneous Time Resolved Fluorescence (HTRF), indicating that multiple SARS-CoV-2 spike protein specific binding proteins were successfully selected. For example, the ankyrin repeat domains of SEQ ID NOs: 1 to 11 constitute amino acid sequences of selected binding proteins comprising an ankyrin repeat domain with binding...

example 2

ng Assays

[0412]Surface plasmon resonance (SPR) assays were used to determine the binding affinity of the binding proteins of the invention to the spike protein of SARS-CoV-2.

[0413]All SPR data were generated using a Bio-Rad ProteOn XPR36 instrument with PBS-T (0.005% Tween20) as running buffer. A new neutravidin sensor chip (NLC) was air-initialized and conditioned according to Bio-Rad manual.

[0414]Mono-domain DARPin proteins: In-house chemically biotinylated (via lysines) SARS-CoV-2 Spike Protein (Sino Biologics, cat. 40589-V08B1, Lot MF14MA0701) was captured to ˜3400 RUs (30 ug / ml, 30 ul / min, 300s). Two buffer injections (100 ul / min, 60s) followed by two 12.5 mM NaOH regeneration steps (100 ul / min, 18s) were applied before the first injections. Mono-domain DARPin proteins were injected (at 50 / 16.7 / 5.6 / 1.9 / 0.6 nM (or at 16.7 / 5.6 / 1.9 / 0.6 nM for SEQ ID NO: 9 and 10)) for 180s at 100 ul / min for association and dissociation was recorded for 3600s (at 100 ul / min). The ligand was regener...

example 3

l Screening

[0421]This Example describes functional screening of mono-domain and multi domain proteins using the SARS-CoV-2 VSV pseudotype virus assay. The results of this assay are provided in FIGS. 5 to 8.

[0422]Infection inhibition was assessed using a vesicular stomatitis virus (VSV) pseudovirus assay (psVSV), where the glycoprotein of VSV was replaced by the Wuhan variant of the SARS-CoV-2 spike glycoprotein tagged with an enhanced green fluorescent protein (EGFP) and firefly luciferase (LUC). Inhibition of infection following addition of 1 nM, 10 nM, or 100 nM of candidate was measured by simple quantification of EGFP and LUC activity (see Torriani, G. et al., Virology 531, 57-68 (2019)).

[0423]FIGS. 5 and 7 show pseudotype SARS-CoV-2 virus inhibition at 100 nM of various recombinant binding proteins that bind to a single site on the spike protein (mono-domain DARPin® proteins) and three sites on the spike protein (multi-domain DARPin® proteins), respectively. Shorter bars are in...

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Abstract

The present invention relates to recombinant binding proteins comprising one or more designed ankyrin repeat domains with binding specificity for coronavirus spike proteins, nucleic acids encoding such proteins, pharmaceutical compositions comprising such proteins or nucleic acids, and the use of such proteins, nucleic acids or pharmaceutical compositions in the treatment of coronavirus diseases, particularly diseases caused by SARS-CoV-2.

Description

FIELD OF THE INVENTION[0001]The present invention relates to recombinant binding proteins comprising one or more designed ankyrin repeat domains with binding specificity for coronavirus spike proteins, nucleic acids encoding such proteins, pharmaceutical compositions comprising such proteins or nucleic acids, and the use of such proteins, nucleic acids or pharmaceutical compositions in the treatment of coronavirus diseases, particularly diseases caused by SARS-CoV-2.BACKGROUND OF THE INVENTION[0002]With a positive-stranded RNA genome of 28 to 32 kb, the Coronaviridae are the largest enveloped RNA viruses. Coronaviruses infect many different mammalian and avian species. They are responsible for a variety of acute and chronic diseases of the respiratory, hepatic, gastrointestinal, and neurological systems.[0003]The common cold is an example of a mild form of coronavirus infection. The 2003 SARS outbreak and the 2012 MERS outbreaks were both caused by coronaviruses. SARS-CoV-2 (also ca...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/47A61P31/14
CPCC07K14/47A61K38/00A61P31/14A61K2039/505C07K2317/33C07K2317/35C07K2317/76C07K2317/92C07K2317/94C07K2318/20C07K16/1003C07K16/10C07K2319/00
Inventor AMSTUTZ, PATRICKCALABRO, VALERIE PERRINEWALSER, MARCEL
Owner MOLECULAR PARTNERS AG
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