Benralizumab reduces blood basophil percentage and activation in vitro without eliciting degranulation
Authors: Marta Gil‐Martínez, José Manuel Rodrigo‐Muñoz, Clara Lorente‐Sorolla, Zahara García de Castro, Pablo Mínguez, José Antonio Cañas, Marcela Valverde‐Monge, Jaime Bernaola, Erwin Javier Pinillos‐Robles, Diana Betancor, Mar Fernández‐Nieto, Joaquín Sastre, María Jesús Rodríguez‐Nieto, Victoria del Pozo
Journal: Allergy (2024)

Benralizumab, a humanized afucosylated IgG1 monoclonal antibody directed against the IL-5 receptor (IL5Rα), is effective in severe eosinophilic asthma (SEA). Its mechanism of action is mainly based on antibody-dependent cell-mediated cytotoxicity of eosinophils,1 the main effector cell in SEA, although benralizumab can target any cell expressing IL5Rα, such as basophils.2 However, it remains unknown whether the mechanism of action of benralizumab involves reducing the activity of basophils or induced basophil degranulation and subsequent promotion of allergic reactions. In this study (NTC04742504), we aim to evaluate the effect of benralizumab on basophils from patients with SEA at different treatment time points (i.e., baseline, 8 weeks, 6 months). We further characterize the in vitro effects of benralizumab on the functionality of human basophils and the human basophil cell line KU812, and lastly investigate whether the miARN profile of basophil changes following application of this biological therapy. Patients with SEA were enrolled both before and after benralizumab treatment to evaluate changes in basophil populations, which were detected by flow cytometry. In addition, KU812 cell lines were treated with benralizumab in vitro. See Data S1 for a detailed description of the study materials and methods. The clinical characteristics of the SEA patients who started therapy with benralizumab (n = 10) are shown in Table S1. Briefly, benralizumab produced improvements in FEV1 and FVC% that were most pronounced after 6 months of treatment. At 8 weeks, there was a non-significant increase in FeNO levels, possibly due to the discontinuation of inhaled corticosteroids as symptoms improved with benralizumab. As expected, blood eosinophil percentage was reduced by 99.7% and 99.4% at 8 weeks and 6 months, respectively (p < .05; Figure 1A), and there was a similar reduction in basophil percentage, from 0.3 ± 0.2% at baseline to 0.07 ± 0.07% at 8 weeks and 0.05 ± 0.06% at 6 months of treatment (p < .05; Figure 1A). The total number of ILC2 cells remained unchanged (Figure 1B). The basophil activation test was conducted on the remaining basophils. Despite the intense depletion observed after treatment, there was no change in activation status after stimulation with benralizumab at any stage of treatment (Figure 1C). We confirmed this finding using blood basophils from allergic asthmatics (n = 8), which showed a similar activation pattern with and without benralizumab (Figure 1C). In vitro, benralizumab slightly reduced the percentage of activated KU812 cells (p < .05; Figure 1D). This limited suppression could be related to the lower number of IL5Rα.3 However, even modest suppression could have a considerable impact on clinical outcomes if these cells are relevant to the disease or response to treatment, and interactions between various cells and biological processes could amplify this effect. Furthermore, the suppression observed could be sufficient to cause a significant biological effect at higher doses or with prolonged treatment. In an additional finding, benralizumab did not alter histamine release (Figure 1E). Finally, purified blood basophils from 7 paired patient samples at baseline (vb) and 8 weeks (8wk) were selected to perform miRNA-seq, which detected upregulation of hsa-miR-451a after 8 weeks of treatment (Figure 1F). Using DIANA-miRPath, we found that miR-451a regulated ABC transporters (hsa02010; p < .05), mTOR (hsa04150; p < .05), and ubiquitin-mediated proteolysis (hsa04120; p < .05). Our results confirm those of previous studies and further support the efficacy of benralizumab in reducing basophils counts.4 Additionally, this biologic therapy reduces the percentage of activated basophils in vitro without affecting histamine release. As in the report by Sehmi et al.,1 total ILC2 was not reduced; however, the earlier study found that benralizumab reduced IL5Rα+ ILC2. MiR-451a was upregulated in basophils after treatment; an increase was also found in the serum of anaphylactic patients.5 In addition, targeted pathways (i.e., ABC transporters, mTOR, and ubiquitin-mediated proteolysis) have been implicated in either basophil activation or asthma.6, 7 In conclusion, benralizumab provides a dual benefit to SEA patients with allergy by reducing not only eosinophils but also basophil percentage and activation and is safe to administer. Moreover, increased levels of hsa-miR-451a are seen in the basophils of treated patients. These findings in atopic SEA patients could be considered complementary mechanisms of symptoms improvement mediated by benralizumab, the only biologic that directly affects the basophil population. VdelP conceived of the manuscript and designed the study. MG-M, JMR-M, CL-S, ZG-dC, PM, and JAC performed experiments and data analysis. MG-M, JMR-M, CL-S, and VdelP edited and wrote the manuscript. MV-M, JB, EJP-R, DB, MF-N, JS, and MJR-N recruited patients and obtained samples and clinical data. All authors contributed to the article and approved the submitted version. The authors thank the ISCIII – Instituto de Salud Carlos III and are grateful for co-funding received from the European Union, FIS (Fondo de Investigación Sanitaria – Spanish Health Research Fund) grants PI21/00896 and FI19/00067; Miguel Servet Program (CP23/00017); CIBER de Enfermedades Respiratorias (CIBERES); BASEAS (Basophils in Eosinophilic Asthma) Study, Study Code ESR-20-20764 AstraZeneca Farmacéutica Spain, S.A.; and FEDER (Fondo Europeo de Desarrollo Regional) funds. The authors would also like to thank all the patients for their voluntary participation as well as all technical and nursing staff involved in the project (Manuela Garcia del Potro, Erica Aguado Wakui, Esther Gamella Álvarez, María Remedios Marquina Valero, Almudena Batanero Rodríguez and Raquel García Latorre). The authors also acknowledge Oliver Shaw for the editorial support. This work was supported by ISCIII – Instituto de Salud Carlos III and co-funded by the European Union, FIS (Fondo de Investigación Sanitaria – Spanish Health Research Fund) grants PI21/00896 and FI19/00067; Miguel Servet Program (CP23/00017); CIBER de Enfermedades Respiratorias (CIBERES); BASEAS (Basophils in Eosinophilic Asthma) Study, Study Code ESR-20-20764 AstraZeneca Farmacéutica Spain, S.A.; and FEDER (Fondo Europeo de Desarrollo Regional) funds. JMR-M reports receiving payments for lectures and educational events from AstraZeneca and GSK. MV-M reports receiving payments for lectures and support for attending meetings and/or travel by AstraZeneca, GSK, Gebro and Organon S.A.; as well as having received grant support for research from Organon S.A. D-B reports having received a contract from Instituto de Salud Carlos III (Rio Hortega Research Contract). J-S reports having served as a consultant to SANOFI, ABBIES, and NOVARTIS; having been paid lecture fees by SANOFI, GSK, and Faes Farma; support for attending meetings and/or travel by SANOFI; and unpaid Leadership or fiduciary role in other board, society, committee, or advocacy groups in SEAIC, AAAAI, and EAACI as well as having received grant support for research from SANOFI. MJR-N reports receiving grant support for research from AstraZeneca, and receiving payments for lectures by AstraZeneca and SANOFI. VdP has received honoraria (advisory board, speaker) and/or institutional grant/research support from AstraZeneca and GSK and unpaid Leadership or fiduciary roles in committees of EAACI. The rest of authors declare no conflicts of interest. The study protocol was approved by the Ethics Committee of hospital Fundación Jiménez Díaz, and the study was conducted in accordance with the principles set forth in the Declaration of Helsinki. Signed informed consent was obtained from all patients. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.