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Publication Number
US-11365403-B2
Publication Date
2022-06-21
Expiration Date
Abstract
The present invention relates to an isolated polypeptide having beta-lactamase activity and nucleic acid sequences encoding the polypeptide. The isolated polypeptide of the invention is a VIM-2 variant with improved properties such as improved protease stability, stability in intestinal medium, improved activity against one or more antibiotics, improved specific activity and/or improved production in a host cell.
Core Innovation
VIM-2 subclass B1 metallo-β-lactamase variants are provided as polypeptides having beta-lactamase activity, comprising an amino acid sequence having at least 90% identity to SEQ ID NO:1. The variants are engineered by substitutions at positions 10 and/or 22 and/or 34 and/or 130, where position numbering corresponds to SEQ ID NO:1 and the substitutions include V10A, Q22H or Q22N, Q34R, and E130D. Optional variants include V1M.
The disclosed engineering is associated with improved protease/intestinal stability, including thermal stability, and with improved beta-lactamase activity compared with wild-type VIM-2 of SEQ ID NO:1. The variants are reported to broaden β-lactam hydrolysis and to improve kinetic/catalytic performance, including kcat, Km, and kcat/Km, as well as increasing production yield in host cells.
The patent further provides a therapeutic concept for degrading residual β-lactam antibiotics in gut regions using orally deliverable compositions/hosts and a β-lactam antibiotic-sensitive kit-of-parts. Documented assay outcomes include intestinal extract stability, enhanced activity in intestinal medium and toward carbapenems, and higher residual hydrolytic activity after incubation across piperacillin, imipenem, meropenem, and ceftriaxone.
Claims Coverage
The claim set centers on SEQ ID NO:1-based VIM-2 polypeptides with improved stability and beta-lactamase activity, together with orally administrable compositions, kits-of-parts, therapeutic treatment, and additional embodiment constraints such as truncations and recombinant host cells.
Polypeptide with SEQ ID NO:1 identity and specified position substitutions
A polypeptide having beta-lactamase activity comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, with substitutions at positions 10 and/or 22 and/or 34 and/or 130, including V10A, Q22H or Q22N, Q34R, and E130D, and improved stability and beta-lactamase activity compared with wild-type VIM-2 of SEQ ID NO:1.
Orally administrable intestinal-release composition
A composition that is orally administrable and capable of releasing a polypeptide in a desired part of the intestine.
Beta-lactam antibiotic-sensitive kit-of-parts
A kit-of-parts including the polypeptide and a beta-lactam antibiotic that is sensitive to the polypeptide, for separate, sequential, or simultaneous administration.
Therapeutic treatment of a bacterial infection with a sensitive beta-lactam antibiotic
A method of treating a bacterial infection in a subject by administering a therapeutically effective amount of a polypeptide, a composition, a kit-of-parts, or an isolated recombinant host cell, together with a beta-lactam antibiotic that is sensitive to the administered polypeptide.
Polypeptide variants with N-terminal and/or C-terminal truncation
A polypeptide that includes a truncation at its N-terminal and/or C-terminal end compared to the sequence in SEQ ID NO:1.
Isolated recombinant host cell containing the recited nucleic acid construct
An isolated recombinant host cell that comprises the nucleic acid construct recited in the dependent nucleic-acid claim.
Coverage is built around VIM-2 polypeptide variants with specified substitutions and improved stability and beta-lactamase activity, with additional claims directed to compositions, kits-of-parts, treatment, truncations, and host-cell embodiments.
Stated Advantages
Improved protease/intestinal stability, including thermal stability.
Broadened β-lactam hydrolysis.
Improved kinetic/catalytic performance, including kcat, Km, and kcat/Km.
Increased production yield in host cells.
Improved residual hydrolytic activity in intestinal medium and enhanced activity toward carbapenems.
Documented Applications
Degrading residual β-lactam antibiotics in gut regions, including jejunal/ileal/caecal/colon, using orally deliverable compositions/hosts and a beta-lactam antibiotic-sensitive kit-of-parts.
Treating bacterial infections in a subject by administering the polypeptide/composition/kit-of-parts/isolated recombinant host cell together with a beta-lactam antibiotic that is sensitive to the administered polypeptide.
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