Infections caused by the opportunist pathogens Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) are of particular concern due to their resistance to multiple drugs (MDR). These pathogens can lead to severe infections in patients with cystic fibrosis (CF) and those who are immunocompromised. Chronic lung infections caused by these bacteria remain problematic to CF patients because of their difficult eradication using the currently available treatments. Moreover, these infections are associated with a faster decline in lung function, increased morbidity and mortality rates. So, currently, novel treatment approaches are being pursued to decrease the use of antibiotics. With this aim, passive immunotherapy has been revisited for MDR pathogens infection prevention and/or treatment. In particular, monoclonal antibodies (mAbs) have been studied as neutralizers of bacterial virulence factors, being regarded as alternative or complement to antibiotic therapy, resulting in faster infection resolution and shorter stays in intensive care units (ICU) as well as reduction of morbidity, mortality and health care costs. However, their use has some drawbacks, such as the large size of mAbs that can lead to inaccessibility to some epitopes and relatively high production costs. Single-domain antibodies (sdAbs) offer several advantages, including smaller size, a larger number of accessible epitopes, relatively low production costs and improved robustness. Recently, immunoconjugates using mAbs or sdAbs as scaffolds have been explored as potential therapeutics for infectious diseases and as alternatives to antibiotics, directing unspecific antimicrobials to the pathogens. One group of antimicrobials extensively studied are the antimicrobial peptides (AMPs) that have the advantage of being abundant in nature. However, several drawbacks have limited their use as antimicrobials, such as their low stability, low specificity, and off-target toxicity. To overcome these limitations, antibody conjugates with AMPs have been studied and shown to increase the stability and specificity of the antimicrobial agent. However, this therapeutic approach against Bcc and P. aeruginosa infections is poorly explored. Surface-exposed antigens are important pathogenicity-related molecules involved in the interactions between hosts and microorganisms, being useful as targets for the development of therapeutic antibodies. Recently, our research group identified the protein BCAL2645 from B. cenocepacia and their orthologues in P. aeruginosa and B. multivorans as a potential target for the development of new therapeutic antibodies that interfere with host-pathogen interactions and neutralize the infections caused by these bacteria. In the present proposal, we aim to develop a sdAb anti-BCAL2645 with the ability to target and neutralize P. aeruginosa and Bcc infections. Additionally, to study this sdAb as a scaffold to generate an antibody-drug conjugate (ADC) combined with a synthetic antimicrobial peptide. The directed cytotoxicity of the new ADC against P. aeruginosa and Bcc infections will be assessed. The research team of the project has all the different expertise required for the achievement of each task delivery proposed. The BSRG/iBB research team has long-term experience in Bcc and P. aeruginosa bacteria biology and their interaction with the host. Lately, the BSRG/iBB research team has been focusing their research on the identification and study of bacterial targets for the development of immunotherapeutic approaches against these bacterial species. Dr. Frederico Aires da Silva (CIISA-FMV) will reinforce the project team with expertise in sdAb and ADC production, purification and characterization. Dr. Marilia Mateus (2BRG/iBB) will reinforce the project with expertise in protein overexpression and purification methods. In summary, this proposal is expected to contribute to the development of a new immunotherapeutic approach using sdAbs as a scaffold to generate ADCs combined with a synthetic antimicrobial peptide to combat earlier established Bcc and/or P. aeruginosa infections, allowing the improvement of their quality of life and lifetime expectancy. This project is aligned with the “One Health” approach from WHO and the United Nations Sustainable Development Goal 3 “Ensure healthy lives and promote well-being for all at all ages”.