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Rationale Monoclonal antibody therapies are used to treat severe asthma. Mepolizumab was the first anti-cytokine therapy introduced, and it targets IL-5. It effectively reduces steroid use and exacerbations in some severe asthma patients but approximately one third of treated patients do not havea clinically significant reduction inexacerbations and oral steroid use at 12 months post-treatment. An early biomarker that was predictive of clinical response at one year would reduce patient burden, improve management and have associated cost saving.Cell free DNA (cfDNA) are DNA fragments released into bodily fluids carrying cell/tissue-specific epigenetic patterns called DNA methylation (DNAm) that reflect their origin. cfDNAm offers tissue specific specificity to systemic samples. We have shown DNAm changes in the nasal epithelium after Mepolizumab treatment [PMID: 38386780] and distinct plasma cfDNAm profiles between asthmatic and non-asthmatic individuals.Objective To identify cfDNAm signatures that differ between Mepolizumab responders (R) and non-responders (NR) pre-treatment and after 3 months of treatment.Methods cfDNA was extracted (Maxwell® RSC ccfDNA Plasma Kit) from 1 ml blood plasma of 14 asthmatic individuals (9 R; 5 NR identified at 12 months) at baseline and 3 months of Mepolizumab treatment [part of Investigating Poor Response to Monoclonal therapy in Asthma study]. DNAm was profiled using the Illumina EPIC Array. Linear regression assessed differential cfDNAm between R and NR.Results A single DNAm locus was identified with a significant (Benjamini-Hochberg p<0.05) decrease (~8%) in methylation between baseline and 3 months among Mepolizumab responders, while non-responders exhibited an increase (~4%). A 213bp region around this locus with 4 adjacent loci was identified to be evaluated as potential early response biomarker. Linear regression analysis within each time point identified DNAm changes (pre-treatment = 1838; 3 months post treatment =1283, nominal p<0.001) between R and NR. The two most significant and consistent loci have been selected to evaluate as potential biomarkers.Conclusion Plasma cfDNAm signatures have been identified for further investigation as early predictors of patient response to Mepolizumab. Evaluation of their potential as biomarkers using pyrosequencing in a blinded blood plasma sample cohort of Mepolizumab treated severe asthmatic individuals is ongoing.