Back to results
US 2026/0193328 A1Published application
Thermostable binding scaffolds
AssigneeNectagen, Inc.
InventorsRichard J. SUDERMAN, David M. CHAO
Life of this patent
Estimating the term…
Abstract
The present invention features thermostable protein binding scaffolds containing framework regions and variable loop regions that can be mutagenized to bind a desired target. The scaffolds are derived from the Carbohydrate Binding Module Family 32 (CBM32) protein domain of Clostridium perfringens hyaluronidase (NagH). The robust framework of the scaffolds described herein allows for the development of custom, high performance affinity chromatography resins compatible with the harsh conditions of process-scale applications that can be adaptable to a wide diversity of target substrates.
Claims84
A protein scaffold comprising the structure:
- wherein:
- F1-F9 correspond to framework regions 1-9;
- L1-L8 correspond to loop regions 1-8;
- A and B are each independently, absent or comprise at least one amino acid;
- F1 comprises the sequence of:
- (T/S)-LI-(H/R)-(T/S)-(P/E)-(G/S)-W (SEQ ID NO:
- 4) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 4;
- L1 is absent or comprises at least one amino acid;
- F2 comprises the sequence of:
- G-(S/N/T)-E-(A/S)-(D/N/S/A)-LLDGDD-(S/N/T)-TGV-(E/W/A)-Y (SEQ ID NO:
- 5) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 5;
- L2 is absent or comprises at least one amino acid;
- F3 comprises the sequence of:
- S-(L/V)-AGEFIGLDLG (SEQ ID NO:
- 6) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 6;
- L3 is absent or comprises at least one amino acid;
- F4 comprises the sequence of:
- G-(I/V)-(H/R/Y/N)-FVIG-(A/K/R)-(D/N) (SEQ ID NO:
- 7) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 7;
- L4 is absent or comprises at least one amino acid;
- F5 comprises the sequence of:
- DKW-(T/N/S)-(R/K)-F-(R/K)-LEYS (SEQ ID NO:
- 8) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 8;
- L5 is absent or comprises at least one amino acid;
- F6 comprises the sequence of:
- WTTI-(R/K/H/Q)-EYD-(H/K/R/Q) (SEQ ID NO:
- 9) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 9;
- L6 is absent or comprises at least one amino acid;
- F7 comprises the sequence of:
- (Q/K)-DVI-(D/E)-E-(D/S)-F (SEQ ID NO:
- 10) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 10;
- L7 is absent or comprises at least one amino acid;
- F8 comprises the sequence of:
- (Q/K/R)-YIRLTNLE (SEQ ID NO:
- 11) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 11;
- L8 is absent or comprises at least one amino acid;
- and
- F9 comprises the sequence of:
- LTFSEFA-(I/V)-VS (SEQ ID NO:
- 12) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 12;
- and
- wherein the protein scaffold comprises at least one mutation selected from the group consisting of N807X, S809X, R812X, S813X, E814X, S815X1, D818X, N822X, N825X, N832X, W836X, K857X, E858X, I859X, K860X2, L861X, D862X, R865X, K870X, N871X, N880X, K881X, K883X, N890X, K897X, K901X, K908X, E912X, S914X, and K922X3 relative to SEQ ID NO:
- 1, wherein:
- X is any amino acid except the amino acid in the equivalent position in SEQ ID NO:
- 1;
- X1 is any amino acid except R or S;
- X2 is any amino acid except P or K;
- and
- X3 is any amino acid except R or K.
55 more elements
A protein scaffold comprising a polypeptide having at least 80% sequence identity to SEQ ID NO:
- 3.
A protein scaffold comprising framework regions and loop regions, the protein scaffold comprising at least 7 framework regions from the following structure:
- wherein:
- F1-F9 correspond to framework regions 1-9;
- L1-L8 correspond to loop regions 1-8 that are each independently, absent or comprises one or more amino acids;
- A and B are each independently, absent or comprise at least one amino acid;
- F1 comprises the sequence of:
- (T/S)-LI-(H/R)-(T/S)-(P/E)-(G/S)-W (SEQ ID NO:
- 4) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 4;
- F2 comprises the sequence of:
- G-(S/N/T)-E-(A/S)-(D/N/S/A)-LLDGDD-(S/N/T)-TGV-(E/W/A)-Y (SEQ ID NO:
- 5) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 5;
- F3 comprises the sequence of:
- S-(L/V)-AGEFIGLDLG (SEQ ID NO:
- 6) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 6;
- F4 comprises the sequence of:
- G-(I/V)-(H/R/Y/N)-FVIG-(A/K/R)-(D/N) (SEQ ID NO:
- 7) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 7;
- F5 comprises the sequence of:
- DKW-(T/N/S)-(R/K)-F-(R/K)-LEYS (SEQ ID NO:
- 8) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 8;
- F6 comprises the sequence of:
- WTTI-(R/K/H/Q)-EYD-(H/K/R/Q) (SEQ ID NO:
- 9) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 9;
- F7 comprises the sequence of:
- (Q/K)-DVI-(D/E)-E-(D/S)-F (SEQ ID NO:
- 10) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 10;
- F8 comprises the sequence of:
- (Q/K/R)-YIRLTNLE (SEQ ID NO:
- 11) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 11;
- and
- F9 comprises the sequence of:
- LTFSEFA-(I/V)-VS (SEQ ID NO:
- 12) or a sequence having one or two amino acid insertions, deletions, substitution mutations, or a combination thereof relative to SEQ ID NO:
- 12.
38 more elements
Dependencies are inferred from the claim text.