Health care authorities are calling for new antibacterial therapies to cope with the global emergence of antibiotic-resistant bacteria. Bacteriophage-encoded lysins are a unique class of antibacterials with promising (pre)clinical progress. Custom engineering of lysins allows for the creation of variants against potentially any bacterial pathogen. We here present a high-throughput hit-to-lead development platform for engineered lysins. The platform is driven by VersaTile, a new DNA assembly method for the rapid construction of combinatorial libraries of engineered lysins. We constructed approximately 10,000 lysin variants. Using an iterative screening procedure, we identified a lead variant with high antibacterial activity against Acinetobacter baumannii in human serum and an ex vivo pig burn wound model. This generic platform could offer new opportunities to populate the preclinical pipeline with engineered lysins for diverse (therapeutic) applications.

Endolysins and their derivatives have emerged in recent years as a novel class of antibacterials, which have now entered the clinical phases. Their rapid mode-of-action and proteinaceous nature differentiates them from any other class of antibiotics. A key feature of endolysins is their modularity and the opportunities that emerge thereof to customize properties such as specificity, activity, stability and solubility. Extensive protein engineering efforts have expanded the activity spectrum to (pan)drug-resistant Gram-negative bacteria or have improved the activity against Gram-positive pathogens. In addition, specific cell wall binding domains derived from endolysins are exploited for the development of diagnostics.

The therapeutic potential of phages has been considered since their first identification more than a century ago. The evident concept of using a natural predator to treat bacterial infections has, however, since then been challenged considerably. Initially, the vast success of antibiotics almost eliminated the study of phages for therapy. Upon the renaissance of phage therapy research, the most provocative and unique properties of phages such as high specificity, self-replication and co-evolution prohibited a rapid preclinical and clinical development. On the one hand, the typical trajectory followed by small molecule antibiotics could not be simply translated into the preclinical analysis of phages, exemplified by the need for complex broad spectrum or personalized phage cocktails of high purity and the more complex pharmacokinetics. On the other hand, there was no fitting regulatory framework to deal with flexible and sustainable phage therapy approaches, including the setup and approval of adequate clinical trials. While significant advances are incrementally made to eliminate these hurdles, phage-inspired antibacterials have progressed in the slipstream of phage therapy, benefiting from the lack of hurdles that are typically associated with phage therapy. Most advanced are phage lytic enzymes that kill bacteria through peptidoglycan degradation and osmotic lysis. Both phages and their lytic enzymes are now widely considered as safe and have now progressed to clinical phase II to show clinical efficacy as pharmaceutical. Yet, more initiatives are needed to fill the clinical pipeline to beat the typical attrition rates of clinical evaluation and to come to a true evaluation of phages and phage lytic enzymes in the clinic.