Engineered methionine gamma lyase variants
Inventors
Franklin, Kierra Aleece • Galanie, Stephanie Sue • Huisman, Gjalt W. • Kruse, Nikki D. • McCluskie, Kerryn • MITCHELL, VESNA • Teadt, Leann Quertinmont • Vallieu, Kristen Jean
Assignees
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Publication Number
US-12110521-B2
Publication Date
2024-10-08
Expiration Date
Abstract
The present invention provides engineered methionine gamma lyase polypeptides and compositions thereof. The engineered methionine gamma lyase polypeptides have been optimized to provide improved thermostability, protease stability, and stability under a range of pH conditions, including acidic (pH<7) conditions. The present invention also relates to the use of the compositions comprising the engineered methionine gamma lyase polypeptides for therapeutic purposes.
Core Innovation
The invention relates to engineered recombinant methionine gamma lyase polypeptides and biologically active fragments defined by polypeptide sequence identity to reference sequences, including at least 85% sequence identity to the reference sequence of SEQ ID NO: 1488. The recombinant methionine gamma lyase comprises at least one specified amino-acid substitution at positions relative to the reference sequence of SEQ ID NO: 2, including 189I/L/M/P/S, 69I/R/W, 237K/A/G/H/L/R/T/Y, 236A/C/R, or 341F, or combinations thereof.
The disclosure further provides extensive enumerations of engineered methionine gamma lyase polypeptide sequence identity thresholds and substitution position patterns relative to SEQ ID NO: 2 and other SEQ IDs, including substitution-set position patterns. It also includes functional fragments and truncations by limited amino acids, with retained activity stated for functional fragments and optional removal of N-terminal initiating methionine/formylmethionine.
The engineered variants are characterized by improved properties including increased tolerance to acid pH, increased thermotolerance, increased tolerance to pH 5.2, increased tolerance to at least one protease, increased activity relative to a reference methionine gamma lyase having the sequence of SEQ ID NO: 2, and improved catalytic activity compared to reference methionine gamma lyase sequences. The document further connects these enzymes to pharmaceutical compositions and to treatment of homocystinuria, with treatment objectives directed to reducing plasma methionine and plasma homocysteine.
Claims Coverage
The independent claim coverage centers on recombinant methionine gamma lyase polypeptides defined by sequence identity to SEQ ID NO: 1488 and substitutions at positions relative to SEQ ID NO: 2. The claim set includes 2 core inventive features and dependent claims that tighten sequence identity thresholds, expand or specify substitution choices and substitution sets, and cover pharmaceutical compositions containing the recombinant enzyme.
Recombinant methionine gamma lyase with reference sequence identity and position-relative substitutions
A recombinant methionine gamma lyase comprising a polypeptide sequence comprising at least 85% sequence identity to the reference sequence of SEQ ID NO: 1488, wherein the polypeptide sequence comprises at least a substitution 189I/L/M/P/S, 69I/R/W, 237K/A/G/H/L/R/T/Y, 236A/C/R, or 341F, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 2.
Pharmaceutical composition including the recombinant methionine gamma lyase
A pharmaceutical composition comprising the recombinant methionine gamma lyase of claim 1.
Overall claim coverage is focused on recombinant methionine gamma lyase polypeptides defined by sequence identity to SEQ ID NO: 1488 and by amino-acid substitutions at positions defined relative to SEQ ID NO: 2, with dependent claims refining identity thresholds and substitution sets. The claim family also extends to pharmaceutical compositions containing the recombinant enzyme.
Stated Advantages
Increased tolerance to acid pH.
Increased thermotolerance.
Increased tolerance to pH 5.2.
Increased tolerance to at least one protease.
Increased activity relative to a reference methionine gamma lyase having the sequence of SEQ ID NO: 2.
Improved catalytic activity compared to reference methionine gamma lyase sequences.
More resistant to proteolysis and bile salt presence.
More thermostable variants.
Stable at acidic pH values including pH 5.0/5.2.
Suppression of serum homocysteine.
Suppression of plasma methionine.
Strong suppression of plasma methionine in cynomolgus monkeys after methionine spikes.
Does not affect phenylalanine or tyrosine in the reported cynomolgus monkey study context.
Dose-dependent suppression of methionine and homocysteine (tHcy) spikes in mouse Tg-1278T Cbs−/−.
Repeat dosing effects on methionine AUC and spike magnitude are reported.
Treating homocystinuria by reducing plasma and/or serum methionine and homocysteine.
Documented Applications
Treating homocystinuria (HCU) by administering a pharmaceutical composition containing the engineered recombinant methionine gamma lyase, with objectives directed to reducing plasma methionine and plasma homocysteine.
Pharmaceutical composition comprising the recombinant methionine gamma lyase.
In vivo characterization in cynomolgus monkeys using peptone- or whey-induced methionine spikes to suppress plasma methionine.
Pharmacodynamics in mouse Tg-1278T Cbs−/− using methionine and homocysteine (tHcy) spikes, including dose-dependent measurements and repeat dosing effects.
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