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EP-4429690-B1 - A CHIMERIC ENDOLYSIN POLYPEPTIDE

EP4429690B1EP 4429690 B1EP4429690 B1EP 4429690B1EP-4429690-B1

Inventors

  • SCHMELCHER, MATHIAS
  • RÖHRIG, Christian Alexander
  • HUEMER, MARKUS
  • Eichenseher, Fritz

Dates

Publication Date
20260513
Application Date
20231222

Claims (13)

  1. An endolysin polypeptide that has lytic activity for Staphylococcus, said endolysin polypeptide comprising a polypeptide, wherein the amino acid sequence of the polypeptide has at least 98% sequence identity with SEQ ID NO: 1; wherein the endolysin polypeptide has at least 10% enhanced lytic activity at 37°C for Staphylococcus in human serum as measured using a quantitative killing assay, compared to: - the endolysin with the amino acid sequence as set forward in SEQ ID NO: 2, and/or - the endolysin with the amino acid sequence as set forward in SEQ ID NO: 3; and wherein the M23 endopeptidase domain and the CHAP domain in the endolysin polypeptide are separated by a linker that comprises at least 13 amino acids.
  2. An endolysin polypeptide according to claim 1, wherein the amino acid sequence of the endolysin polypeptide has at least 98% sequence identity with SEQ ID NO: 1.
  3. An endolysin polypeptide according to claim 1, wherein the amino acid sequence of the endolysin polypeptide is as set forward in SEQ ID NO: 1.
  4. A polynucleotide encoding the endolysin polypeptide according to any one of claims 1 to 3.
  5. A nucleic acid construct comprising the polynucleotide according to claim 4.
  6. An expression vector comprising the nucleic acid construct according to claim 5.
  7. A host cell comprising the polynucleotide according to claim 4, the nucleic acid construct according to claim 5 or the expression construct according to claim 6.
  8. A method for the production of an endolysin polypeptide according to any one of claims 1 to 3, comprising: - culturing a host cell according to claim 7 under conditions conducive to the production of the endolysin polypeptide, - optionally isolating and purifying the endolysin polypeptide from the culture broth, and - optionally freeze-drying or spray-drying the endolysin polypeptide.
  9. A composition comprising an endolysin polypeptide according to anyone of claims 1 to 3, or a polynucleotide according to claim 4, or a nucleic acid construct according to claim 5, or an expression construct according to claim 6, or a host cell according to claim 7.
  10. A composition comprising an endolysin polypeptide according to any one of claims 1 to 3, or a polynucleotide according to claim 4, or a nucleic acid construct according to claim 5, or an expression construct according to claim 6, or a host cell according to claim 7, further comprising an excipient acceptable for cosmetics.
  11. A pharmaceutical composition comprising an endolysin polypeptide according to any one of claims 1 to 3, or a polynucleotide according to claim 4, or a nucleic acid construct according to claim 5, or an expression construct according to claim 6, or a host cell according to claim 7, said pharmaceutical composition further comprising a pharmaceutically acceptable excipient.
  12. A composition according to claim 9, 10 or 11, further comprising an additional active ingredient.
  13. A composition according to claim 9, 11 or 12, for use as a medicament, preferably for use as a medicament in the treatment of a condition associated with infection with a Staphylococcus.

Description

Field of the invention The invention relates to the field of medicine, specifically to the field of treatment of conditions associated with Staphylococcus infection. The invention relates to a novel endolysin polypeptide specifically targeting a bacterial Staphylococcus cell. The invention further relates to said endolysin polypeptide for medical use, preferably for treating an individual suffering from a condition associated with Staphylococcus infection. Background of the invention Widespread dissemination of antimicrobial resistance (AMR) genes among pathogenic bacteria has resulted in a health crisis of staggering proportions, with little or no treatment options for those affected and expectations that this problem will become more profound over time. A recent study analyzing the global burden of AMR in 2019 concludes that it has become a leading cause of death, with 1.27 million deaths directly attributable to resistance and many more AMR-associated deaths (ARC., 2022). This is in line with the UK government sponsored Review on Antimicrobial Resistance which estimates that the global death toll caused by AMR could result in an annual 10 million deaths by 2050. The WHO has identified 6 pathogens causing the majority of fatalities due to resistance: Escherichia coli, followed by Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Methicillin resistant S. aureus alone was directly responsible for 100.000 deaths in 2019 (ARC., 2022). Staphylococcal bloodstream infections (SBIs) in particular present an enormous challenge for adequate treatment. Permanent S. aureus colonization of the anterior nares in a substantial part of the population (~30%) results in opportunities for the bacteria to enter the bloodstream with potentially devastating consequences. Sepsis and septic shock occur in a significant number of S. aureus bloodstream infection cases, but endocarditis and other deep-tissue infections may result (Song et al., 2020). Coagulase negative staphylococci, most notably S. epidermidis are also a frequent cause of SBIs. Having fewer virulence genes than S. aureus, SBIs caused by S. epidermidis generally presents as subacute or chronic but can disseminate to many parts of the body. Biofilm production is a common feature of many S. epidermidis strains and colonization of implanted devices such as intravascular devices, cerebrospinal fluid shunts, intraocular lenses, prosthetic joints and heart valve replacements frequently occurs. Treatment of established SBIs requires antimicrobials although removal of affected medical devices is recommended. Methicillin resistance among strains of both bacteria is becoming more prevalent, reducing the number of antibiotic treatments available. Vancomycin and linezolid treatment potentially have serious side effects and cases that display increased minimal inhibitory concentration and even complete resistance to these compounds are becoming more frequent. In fact, multidrug resistance has been observed in 70-85% of nosocomial strains of S. epidermidis (Kleinschmidt et al., 2015). There is therefore an urgent need for novel antimicrobial compounds to combat these infections. Peptidoglycan hydrolases (PGHs) can cleave specific bonds within the peptidoglycan (PG) network of bacteria and have been shown to be active against biofilms. Their high lytic activity makes PGHs potent anti-staphylococcal agents. Endolysins are highly specific, phage-derived PGHs, active against both drug-sensitive and resistant bacteria (Schmelcher et al. 2012). As potential alternatives to antibiotics, they have undergone investigations in vitro, in vivo and are under trial in several clinical studies (Kashani et al. 2017). PGHs of Staphylococci regularly display a domain-like architecture, consisting of enzymatically active domains (EADs) and cell-wall-binding domains (CBDs). The high specificity of staphylococcal PGHs may be attributed to their CBDs, which regularly feature an SH3b-fold. The structures of staphylococcal endolysin SH3b domains have been solved and display great homology to the bacteriocins lysostaphin (LST) and ALE1, suggesting a common recognition site in the PG. The EADs are more diverse and can be grouped according to their structure and cleavage site within the PG. Cysteine, histidine dependent amidohydrolase/peptidase (CHAP) domains are frequently found in staphylolytic endolysins, for example in phage Twort or phage K (Korndörfer et al. 2006). Depending on the CHAP domain present, cleavage can occur at different locations in the PG, including the amide bond of the sugar backbone to the stem peptide and the link of the stem peptide to the peptide cross bridge. Herein, the amidohydrolase/peptidase activity of a CHAP domain is referred to as CHAP activity. M23 domains, have only been found in one endolysin (phage 2638), but are also present in the staphylococcal bacteriocins LST and its homologue ALE1. The