Nicotine degrading enzyme variants
Inventors
KALNIK, Matthew W. • Thisted, Thomas • Stone, Everett • Reed, Charles C. • RODNICK-SMITH, Max
Assignees
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Publication Number
US-11597916-B2
Publication Date
2023-03-07
Expiration Date
Abstract
Described are nicotine-degrading enzyme variants that exhibit increased nicotine-degrading activity and/or decreased immunogenicity relative to the wild-type NicA2 and NOX enzymes, compositions comprising the variants, and methods using them.
Core Innovation
The invention relates to nicotine-degrading enzyme variants derived from the wild-type NicA2 enzyme set forth in SEQ ID NO: 1. The variants maintain nicotine-degrading activity while comprising at least 90% sequence identity to SEQ ID NO: 1 or to SEQ ID NO: 1 having an N-terminal deletion of up to 52 amino acids. The variants include at least one substitution at amino acid positions selected from positions 91, 104, 106, 107, 217, 250, 340, 366, 381, 427, 462, and 463 of SEQ ID NO: 1.
The disclosure further specifies changes in immunogenic T-cell epitopes by comprising a substitution, addition, or deletion in an immunogenic T-cell epitope at an amino acid position selected from positions 74, 77, 78, 80, 262-266, 303, 304, 306, 310, 374, 377, 378, 382, 383, 450-452, and 457 of SEQ ID NO: 1. The variants are configured to maintain nicotine-degrading activity while addressing immunogenic T-cell epitopes and reduced immunogenicity.
The disclosure also provides long-acting forms by fusing a nicotine-degrading enzyme variant to a selected compound selected from albumin-binding peptide, albumin-binding protein domain, human serum albumin, an inert polypeptide, recombinant PEG (XTEN), a homo-amino acid polymer (HAP), a proline-alanine serine polymer (PAS), an elastin-like peptide (ELP), and polyethylene glycol (PEG). In certain embodiments, the long-acting variant is PEGylated, and the variant is provided as a pharmaceutical composition with a pharmaceutically acceptable carrier.
Claims Coverage
The independent claims include three claim sets. Across them, the invention is directed to NicA2-based nicotine-degrading enzyme variants with defined sequence identity relative to SEQ ID NO: 1, optional N-terminal deletion up to 52 amino acids, and at least one substitution at specified residue positions, with dependent coverage for immunogenic T-cell epitope changes, long-acting fusion formats, PEGylation, and pharmaceutical compositions.
Nicotine-degrading enzyme variant with defined sequence identity and substitutions
A nicotine-degrading enzyme variant having nicotine-degrading activity, based on wild-type NicA2 in SEQ ID NO: 1, with at least 90% sequence identity to SEQ ID NO: 1 or to SEQ ID NO: 1 having an N-terminal deletion of up to 52 amino acids, and at least one substitution at amino acid positions 91, 104, 106, 107, 217, 250, 340, 366, 381, 427, 462, and 463.
Nicotine-degrading enzyme variant with at least about 95% identity and substitutions
A nicotine-degrading enzyme variant having nicotine-degrading activity and comprising an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 1 or an N-terminal deletion variant of up to 52 amino acids, with at least one substitution at amino acid positions 91, 104, 106, 107, 217, 250, 340, 366, 381, 427, 462, and 463.
Nicotine-degrading enzyme variant with optional N-terminal deletion and specified substitutions
A nicotine-degrading enzyme variant having nicotine-degrading activity comprising SEQ ID NO: 1 with at least one substitution at one or more amino acid positions selected from 91, 104, 106, 107, 217, 250, 340, 366, 381, 427, 462, and 463, or SEQ ID NO: 1 having an N-terminal deletion of up to 52 amino acids and at least one substitution at the same positions.
The independent claims cover NicA2-based nicotine-degrading enzyme variants that retain nicotine-degrading activity while meeting defined sequence identity thresholds relative to SEQ ID NO: 1, with optional N-terminal deletion up to 52 amino acids and at least one substitution at the listed residue positions. Dependent claim coverage further includes immunogenic T-cell epitope-targeting changes, long-acting fusion or PEGylated formats, and pharmaceutical compositions with pharmaceutically acceptable carriers.
Stated Advantages
Improved catalytic performance of nicotine-degrading enzymes via active-site/aromatic-cage-targeted mutations.
Improved nicotine-degrading activity.
Reduced immunogenicity through T-cell epitope reduction/decreased immunogenicity via epitope engineering.
Long-acting properties through extended half-life formats using fusion to specified albumin-binding or polymer/PEG-like compounds, including PEGylated forms.
Documented Applications
Formulation as pharmaceutical compositions including the nicotine-degrading enzyme variants together with a pharmaceutically acceptable carrier.
Use of long-acting/PEGylated nicotine-degrading enzyme variants fused to albumin-binding, polymer/PEG-like compounds for long-acting delivery.
Treating nicotine addiction and smoking cessation.
Treating nicotine poisoning.
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