The main objective of this project is to develop a specific compound to be used in infections caused by multi-resistant E. coli bacteria in humans, based on the use of EPLEs and as an alternative to the use of the current technology used at medical/hospital level, such as antibiotics.

With the spread of antibiotic resistance in almost all pathogenic bacteria, health authorities have identified the search for new antimicrobial alternatives to antibiotics as a priority at the international level. Among the most promising alternatives at present is phage therapy with endolysins, which prevent the generation of bacterial resistance. This strategy has the advantage of being effective against Gram-negative bacteria, which are particularly complex to eliminate due to their outer membrane, thanks to the genetic modification of the endolysins binding domains. This type of therapy, which Telum Therapeutics has been researching for some years, has not yet been demonstrated in standardised or regulatory trials, which is why it has been identified as an interesting challenge to tackle.

Title: NEW PHAGE THERAPY AGAINST ESCHERICHIA COLI INFECTIONS IN HUMANS.

Identification number: 0011-1365-2020-000278

Execution period: April 2020-June 2022

Project executing entity(ies): Telum Therapeutics  S.L., Universidad de Navarra (UNAV).

This project aims to develop broad-spectrum antibacterial compounds for use as empirical therapy in human infections caused by enterobacteria and Pseudomonas aeruginosa (including multi-resistant strains), based on the use of natural enzymes of phage origin and as an alternative to the use of antibiotics.

The current problem with the emergence of antibiotic-resistant bacteria and the lack of therapies to combat them has led to the urgent need to find new effective treatments for these micro-organisms. This need is particularly urgent in the case of Enterobacteriaceae producing BLEE (extended-spectrum β-lactamases) and carbapenemases, as well as Pseudomonas aeruginosa producing cabapenamases, as warned by the World Health Organisation.

Numerous studies place bacteriophages (phages) and their lytic enzymes as one of the most promising alternatives to conventional antibiotics. Bacteriophages have been detected in places such as soils, rivers and seas that have little or no human contact with humans and/or animals, they are an attractive source of all kinds of bacteria, offering the possibility of isolating them for therapeutic purposes against infections. Such therapies, which Telum Therapeutics has been investigating for a number of years, have not yet been shown to have a proven effect in standardised or regulatory trials, and are therefore identified as an interesting challenge to address.

Title: NEW PHAGE THERAPY AGAINST PSEUDOMONAS AERUGINOSA AND MULTIDRUG-RESISTANT ENTEROBACTERIA.

Identification number: 0011-1365-2020-000149

Execution period: April 2020-June 2022

Project executing entity(ies): Telum Therapeutics  S.L.

Staphylococcus aureus is an opportunistic respiratory tract pathogen that can cause severe generalised infections (bacteraemias) in humans. Treatment of such infections can be difficult, as it is a pathogen that carries resistance to multiple antibiotics. As an alternative, TELUM is developing EPLEs (i.e., synthetic enzymes with antimicrobial power) that could be useful as an alternative to conventional treatments against S. aureus. Therefore, the overall objective of this project is to develop a treatment based on EPLEs compounds against S. aureus infections and its evaluation in preclinical murine models.

As a consequence of the spread of antibiotic-resistant bacteria, the search for new treatments and antimicrobial therapies, alternatives to the use of conventional antibiotics, is a priority for the health sector at national and international level. Among the most promising current alternatives is therapy with bacteriophages or part of their components such as endolysins or virion-associated lysin to treat pathogenic bacterial infections.

The technological scope of the project is broad, ranging from the design of the EPLEs, its production under standardised conditions and pre-formulation, to the in vitro and in vivo evaluation of its efficacy and toxicity, after respiratory or intravenous administration. The project is challenging, with particular complexity in the phases of compound purification, evaluation in animals and formulation for respiratory application.

Title: DEVELOPMENT OF ENZIBIOTICS AGAINST RESPIRATORY INFECTIONS DUE TO STAPHYLOCOCCUS AUREUS.

Identification number: 0011-1365-2022-000281

Execution period: 2 years

Project executing entity(ies): Telum Therapeutics  S.L. Instituto de Agrobiotecnología (IDAB-CSIC)

The main objective of this project is to develop a compound for use in infections caused by Enterococcus faecium, based on the use of natural endolysins.

Faced with the expansion of antibiotic resistance in almost all pathogenic bacteria, health authorities have made the search for new antimicrobial treatment alternatives to antibiotics a priority at international level. The WHO has included Enterococcus faecium in the list of pathogens of special urgency because of its high prevalence in serious infections. Among the most promising alternatives at present are phage therapy with endolysins or virion-associated lysin, which prevent the generation of bacterial resistance, whose results improve notably when recombinant protein engineering is applied, and massive sequencing by non-culture-dependent techniques.

However, although research in this area is showing great potential, no biotech company has so far developed EPLEs against Enterococcus faecium.

Title: DEVELOPMENT OF ENZYBIOTICS TO COMBAT HUMAN INFECTIONS CAUSED BY ENTEROCOCCUS FAECIUM RESISTANT TO VANCOMICIN.

Identification number: CPP2021-009054

Execution period: 3 years

Project executing entity(ies): Telum Therapeutics  S.L. Instituto de Salud Carlos III (ISCIII), Universidad Complutense de Madrid (UCM)

Proyecto CPP2021-009054 financiado por MCIN/AEI/10.13039/501100011033 y por la Unión Europea-NextGenerationEU/PRTR

The project in which the post-doctoral candidate’s work is framed aims to research and develop EPLEs for use in infections caused by Enterococcus faecium.

More specifically, the specific technical objectives to be achieved are as follows:

• Generation of an optimised library of phages, EPLEs and domains effective against Enterococcus faecium to vancomycin (VRE).
• Preventive potential of these EPLEs against faecium infection both in planktonic state (systemic infection) and forming biofilm (chronic infection).
• Therapeutic potential against faecium.
• Effectiveness of combined treatment with drugs currently in use (Vancomycin, Daptomycin, Tigecycline, Oxazolidinone, Linezolid, Quinupristin/dalfopristin) that can be used in a complementary manner, facilitating generalised medical prescription.
• High stability under the conditions of its application as a final drug (pH, temperature and concentration of sugar and salts in the medium).
• EEPLE purity of more than 95%.
• Zero toxicity of the EPLE.

Title: DESIGN AND OPTIMISATION OF EPLEs TO COMBAT HUMAN INFECTIONS CAUSED BY VANCOMYCIN-RESISTANT ENTEROCOCCUS FAECIUM RESISTANT TO VANCOMYCIN.

Identification number: PTQ2021-011910

Execution period: 3 years

Project executing entity(ies): Telum Therapeutics  S.L.

Ayuda PTQ2021-011910 financiada por el MCIN/AEI /10.13039/501100011033

The main objective of this project is to develop a panel of compounds to be used in Cystic Fibrosis (CF) infections caused by the pathogens *Pseudomonas aeruginosa* and *Staphylococcus aureus*, based on the use of natural enzybiotics.

Due to the increasing antibiotic resistance in almost all pathogenic bacteria, health authorities have identified the search for new alternative antimicrobial treatments to antibiotics as a priority on a global scale. The WHO has included *Pseudomonas aeruginosa* and MRSA *Staphylococcus aureus* on its list of critically urgent pathogens due to their high prevalence in severe infections and their significant resistance to the antibiotics currently used in clinical practice. Among the most promising alternatives is phage therapy with lysins or enzybiotics, which avoid the development of bacterial resistance. Results improve significantly when combined with recombinant protein engineering and high-throughput sequencing using non-culture-dependent techniques.

However, although research in this area is showing great potential, no biotechnology company has yet developed enzybiotics capable of killing *P. aeruginosa* and *S. aureus* in real-world tests in CF models.

The technological scope of the project is broad, ranging from the isolation and sequencing of phages and enzybiotics from environmental samples to the in vitro and in vivo evaluation of their efficacy and stability. The project as a whole is a challenge, but it will be overcome thanks to the application of bioinformatics tools developed by the project leader, as well as the expertise of partners in biofilm assays, cell lines, and animal models.

The project represents a significant innovation in the field of human health, as existing treatments for CF are currently designed to control its symptoms—primarily mucus retention and chronic infection—but there is no therapy to directly eliminate the first and second causes of infections in these patients. The market niche targeted by the project is global and vast, considering the significant impact of infections caused by—

Title: DEVELOPMENT OF ENZYBIOTICS TO COMBAT INFECTIONS IN CYSTIC FIBROSIS PATIENTS CAUSED BY THE PATHOGENS PSEUDOMONAS AERUGINOSA AND STAPHYLOCOCCUS AUREUS.

Identification number: CPP2022-009574

Execution period: 2 years (Start year 2024)

Project executing entity(ies): Telum Therapeutics S.L. Instituto de Salud Carlos III (ISCIII), Consejo Superior de Investigaciones Científicas (CSIC)

The ENZINETO-V project is part of the global fight against antimicrobial resistance, a major public health threat worldwide. Ventilator-associated pneumonia (VAP), caused by multidrug-resistant pathogens such as Acinetobacter baumannii, is one of the most severe hospital-acquired infections, associated with high morbidity and mortality rates in intensive care units. The increasing resistance of A. baumannii to conventional antibiotics has drastically reduced available therapeutic options, underscoring the urgent need for new antimicrobial strategies.

Enzybiotics, lytic proteins derived from bacteriophages, represent a promising alternative due to their high specificity and potent activity against multidrug-resistant bacteria, while preserving the beneficial microbiota. This project aims to develop and validate, at the preclinical level, an enzybiotic specifically targeting A. baumannii, thereby addressing a critical unmet need in the treatment of nosocomial infections.

The consortium is led by Telum Therapeutics, an expert in the development of enzybiotic-based therapies, and includes V Labs Analytics, a chemical-pharmaceutical analysis laboratory responsible for analytical method development; the University of Navarra (UNAV), with strong expertise in biomedical research and preclinical development; the Spanish National Research Council (CSIC) – Institute of Agrobiotechnology (IDAB), focused on biotechnology and microbiology research; and the Navarra Health Cluster (NHC), responsible for the dissemination and promotion of the project.

The main goal of the project is to investigate a novel therapeutic approach based on enzybiotics targeting A. baumannii, through the development of two administration routes: intravenous and intranasal. The intravenous route is intended for intubated patients, enabling rapid and effective drug delivery directly into the bloodstream—crucial for critically ill patients. In contrast, the intranasal route is designed for non-intubated chronic patients, offering a less invasive and more convenient option suitable for treatment outside intensive care settings.

Title: DEVELOPMENT OF AN ENZYBIOTIC-BASED BIOTECHNOLOGICAL THERAPY TO FIGHT HOSPITAL-ACQUIRED INFECTIONS

Identification number: 0011-1411-2025-000084

Execution period: 2 years (Start year 2025)

Project executing entity(ies): Telum Therapeutics S.L., V Labs Analytics S.L., Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Navarra (UNAV), Navarra Health Cluster (NHC)

Esta entidad ha recibido una ayuda de Gobierno de Navarra cofinanciada al 40% por el Fondo Europeo de Desarrollo Regional a través del Programa Operativo FEDER 2021-2027 de Navarra.