Elevated human epididymis protein 4 (HE4) levels decreased in patients with CF (pwCF) in response to CFTR-specific drugs and negatively correlated with FEV1% predicted values (ppFEV1). Objectives: Although elexacaftor/tezacaftor/ivacaftor (ETI, Kaftrio®) demonstrates more substantial effectiveness than lumacaftor/ivacaftor (LUM/IVA, Orkambi®) in pwCF, plasma biomarkers have not been used to compare treatment efficacy. Hence, our aim was to correlate the change in HE4 levels and the clinical effects of these CFTR modulators (CFTRm). Methods: Serum HE4 concentrations were measured in a total of 123 pwCF homozygous for the p.Phe508del-CFTR variant before treatment and 1-6 months after either ETI or LUM/IVA administration. A correlation between serum HE4 and ppFEV1 was assessed using the Spearman test. HE4 protein levels were also analyzed in the supernatants of p.Phe508del-CFTR CFBE 41o- cells before and after treatment with these CFTRm, and their direct effect on CFTR function was monitored by the whole-cell patch-clamp technique. Results: Serum HE4 levels were reduced below baseline after 3 months of either ETI or LUM/IVA (mean delta HE4: -38.5 vs. -18.5 pmol/L, respectively) when the mean change of ppFEV1 was 13.6 vs. 1.6% and remained decreased up to 6 months. A significant inverse correlation between HE4 and ppFEV1 was observed in both study cohorts (r = -0.537 and r = -0.575, respectively; p < 0.0001). In agreement with ex vivo results, the effect on p.Phe508del-CFTR was more pronounced by ETI than LUM/IVA in CFBE cells, showing a larger improvement in p.Phe508del-CFTR function and reductions in HE4 levels at 24 h. Conclusions: Serum HE4 negatively correlates with lung function improvement and monitors better drug efficacy in pwCF under ETI than LUM/IVA.

BioISI Biosystems and Integrative Sciences Institute Faculty of Sciences University of Lisboa 1749 016 Lisboa Portugal

Department of Biology and Medical Genetics 2nd Faculty of Medicine Charles University and Motol University Hospital 150 06 Prague Czech Republic

Department of Biophysics and Cell Biology Faculty of Medicine University of Debrecen 4032 Debrecen Hungary

Department of Laboratory Medicine Faculty of Medicine University of Debrecen 4032 Debrecen Hungary

Department of Medical Genetics Faculty of Medicine University of Debrecen 4032 Debrecen Hungary

Department of Pediatrics Albert Szent Györgyi Medical School University of Szeged 6725 Szeged Hungary

Department of Pediatrics Faculty of Medicine University of Pécs 7624 Pécs Hungary

Department of Pulmonology 2nd Faculty of Medicine Charles University and Motol University Hospital 150 06 Prague Czech Republic

National Korányi Institute for Pulmonology 1122 Budapest Hungary

Törökbálint Institute for Pulmonology 2045 Törökbálint Hungary

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