Identification of lysins and derivatives thereof with bactericidal activity against Pseudomonas aeruginosa

Inventors

Schuch, Raymond • INDIANI, CHIARA

Assignees

Aurobac Therapeutics SAS

Abstract

Disclosed are novel lysin polypeptides active against Gram-negative bacteria, particularly P. aeruginosa, pharmaceutical compositions containing them and methods for their use to treat Gram-negative bacterial infections and more generally to inhibit the growth, or reduce the population, or kill Gram-negative bacteria, including without limitation disrupting biofilms formed by such bacteria. Certain of the disclosed lysins have been modified in amino acid sequence compared to that of lysins by replacement of certain charged amino acids with noncharged amino acids and/or by fusion at the N- or C-terminus with antibacterial peptide sequences with or without an intervening linker.

Core Innovation

The document describes bactericidal lysin polypeptides that are active against Gram-negative bacteria, with particular emphasis on Pseudomonas aeruginosa. It identifies native lysin polypeptides and evaluates them against P. aeruginosa in human serum, where human serum inactivation is addressed through engineered derivatives. The engineered derivatives are designed to overcome serum inactivation and enable outer-membrane penetration while retaining lytic activity.

The derivatives are generated by amino acid substitutions and by antimicrobial peptide fusions. Amino acid substitutions include substituting charged residues with noncharged amino acids, including K100D and R116H relative to a lysin polypeptide of GN4 (SEQ ID NO: 3). The document also describes fusing N-terminal or C-terminal antimicrobial peptides to the lysin polypeptides, including optional linkers, to create modified lysin polypeptides, fragments having lytic activity, and variants having lytic activity.

The lysin polypeptides and derivatives are presented as providing in vitro bactericidal activity in human serum, including improvements in MIC and anti-biofilm activity. The document further reports absence of hemolytic activity and includes bactericidal time-kill activity. In addition, it describes synergy with multiple Gram-negative antibiotics in checkerboard assays.

Claims Coverage

The independent claims cover five main categories of subject matter: pharmaceutical compositions, vectors, recombinant expression vectors, isolated polynucleotides, and isolated modified lysin polypeptides. Across these independent claims, the inventive core is the use of modified lysin polypeptides (or encoded/contained forms) defined by selected GN-designated sequences or lytic-active fragments/variants sharing at least 80% sequence identity with GN4 (SEQ ID NO: 3) and comprising K100D and R116H substitutions relative to SEQ ID NO: 3.

Across the independent claims, the document covers modified lysin polypeptides (and encoding/containing embodiments) defined by selected GN-designated sequences or lytic-active fragments/variants with at least 80% sequence identity to GN4 (SEQ ID NO: 3) and the amino acid substitutions K100D and R116H. The claim set further spans pharmaceutical compositions with a pharmaceutically acceptable carrier and nucleic-acid/vector formats, including operatively linked heterologous promoter recombinant expression vectors.

Stated Advantages

Documented Applications