Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Dec 26.
doi: 10.1186/s13046-025-03620-3. Online ahead of print.

Revealing erythropoietin variant EV-3 as novel driving force and immunotherapeutic target in human glioblastoma

Stefania Elena Navone #  1 , Giovanni Marfia #  1   2 , Laura Guarnaccia  1 , Massimiliano Rizzaro  1   3 , Giorgio Fiore  1 , Rolando Campanella  4 , Chiara Gaudino  5 , Daniele Santini  6   7 , Giovanni Andrea Alotta  4 , Monica Rosa Miozzo  8 , Emanuela Barilla  4 , Marco Locatelli  1   3 , Laura Riboni  9
Affiliations
Free article

Revealing erythropoietin variant EV-3 as novel driving force and immunotherapeutic target in human glioblastoma

Stefania Elena Navone et al. J Exp Clin Cancer Res. .
Free article

Abstract

Background: Glioblastoma is the most aggressive primary brain tumor, and, despite intensive studies, remains one of the most fatal malignancy in adult humans. Among multiple onco-promoters produced by glioblastoma cells, erythropoietin was found. However, the presence/function of Epo alternatively spliced variants in cancer remains unexplored. Here, we investigated the expression and role of Epo-variants in glioblastoma, and the therapeutic potential of their targeting through a novel monoclonal antibody (mAb).

Methods: Transcripts and protein levels of Epo-variants in a cohort of human brain tumors were evaluated by RT-PCR, ELISA, and immunohistochemistry. Monoclonal antibodies targeting Epo-Vs were prepared and functionally selected by assaying proliferation, migration, stemness, and angiogenesis in glioblastoma patient-derived cells. Antibody affinity for Epo/Epo-variant was determined by SPR. In vivo toxicity and therapeutic efficacy of the lead antibody were evaluated in GBM mouse models.

Results: We found a significant overexpression of Epo-variant transcripts in tissues and cells from GBM patients. After functional selection of newly-produced antibodies, we identified AND-C4 as the lead one for its potent anti-tumoral properties, absence of anti-erythropoietic effects and of toxicity on human brain cells. AND-C4 exhibited high affinity for the Epo-variant EV-3. We demonstrated that EV-3 was efficiently produced and secreted by glioblastoma cells, particularly by stem cells. EV-3 exerted tumorigenic, angiogenic and immunomodulatory properties, and AND-C4 was effective in antagonizing all these actions. In vivo studies in rodent glioblastoma models revealed that AND-C4 selectively bound to tumor tissue and exhibited significant efficacy on tumor growth and animal survival.

Conclusion: This study represents the first evidence on the presence, origin and pro-tumoral activity of EV-3 in human glioblastoma. Moreover, in vitro and in vivo results revealed AND-C4 as novel and promising anti-glioblastoma immunotherapeutic.

Keywords: Cancer immunotherapy; Cancer stem cells; EV-3; Erythropoietin; Glioblastoma; Hypoxia; Monoclonal antibodies; Tumor microenvironment.

PubMed Disclaimer

Conflict of interest statement

Declarations. Ethics approval and consent to participate: All experiments involving animals were authorized by the Italian Ministry of Health and performed in agreement with the authorized protocol and ethical standards, according to the Declaration of Helsinki. Studies involving human participants were approved by the Ethics Committee of the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy. A written informed consent was obtained from each patient prior to his/her enrollment into the study. Consent for publication: We declare that this manuscript is original, has not been published previously, and is not under consideration for publication in any other journal. The manuscript has been read and approved by all Authors, and no individuals who meet the criteria for authorship have been omitted. All Authors have agreed upon the order in which their names are listed. Competing interests: The authors declare the following conflict of interest: SEN, GM, RC, GAA, EB are co-founders and/or holders of Andremacon S.r.l. SEN, GM, RC, GAA, EB, LR are inventors of a patent application related to antibody use. All other authors declare no competing financial or personal interests that could influence the work reported in this manuscript.

References

    1. Louis DN, Perry A, Wesseling P, Brat DJ, Cree IA, Figarella-Branger D, Hawkins C, Ng HK, Pfister SM, Reifenberger G, Soffietti R, von Deimling A, Ellison DW. The 2021 WHO classification of tumors of the central nervous system: a summary. Neuro Oncol. 2021;23:1231–51.
    1. Ostrom QT, Price M, Neff C, Cioffi G, Waite KA, Kruchko C, Barnholtz-Sloan JS. CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the united States in 2016–2020. Neuro Oncol. 2023;25:iv1–99.
    1. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO. Radiotherapy plus concomitant and adjuvant Temozolomide for glioblastoma. N Engl J Med. 2005;352:987–96.
    1. Grochans S, Cybulska AM, Simińska D, Korbecki J, Kojder K, Chlubek D, Baranowska-Bosiacka I. Epidemiology of glioblastoma Multiforme-Literature review. Cancers. 2022;14:2412.
    1. Broekman ML, Maas SLN, Abels ER, Mempel TR, Krichevsky AM, Breakefield XO. Multidimensional communication in the microenvirons of glioblastoma. Nat Rev Neurol. 2018;14:482–95.

LinkOut - more resources