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ORIGINAL RESEARCH article
Front. Immunol.
Sec. Cancer Immunity and Immunotherapy
Volume 16 - 2025 | doi: 10.3389/fimmu.2025.1498697
Trifunctional antibody-cytokine fusion protein formats for tumortargeted combination of IL-15 with IL-7 or IL-21
Provisionally accepted- University of Stuttgart, Stuttgart, Germany
Cytokines from the common gamma chain receptor family, such as IL-15, IL-21 and IL-7, show promise for cancer immunotherapy and have been incorporated individually into the immunocytokine approach. However, their efficacy as monotherapy is limited. Here, we investigated the molecular design of tumor-directed trifunctional antibody-cytokine fusion proteins for a combinatorial approach of IL-15 with either IL-7 or IL-21. Various fusion proteins differing in antibody format, cytokine composition and arrangement were generated and cooperative cytokine activity assessed in solution and bound to target cells. Comparative analysis revealed that formats with cytokines positioned at the N-and C-termini of the antibody were more effective than those arranged in series. For the former design, cooperative effects were observed with the scFv-based (IL-15+IL-7) trifunctional fusion protein, primarily enhancing the proliferation of naive T cells, while the scFv/Fab-based (IL-15+IL-21) trifunctional fusion proteins enhanced IFN-y release and the cytotoxic potential of T cells. Combining cytokines in the two-in-one molecule approach was principally advantageous when bound to target cells. Greater potency in inducing JAK-STAT pathway activation highlighted the importance of cytokine colocalization for cooperative receptor activation. Compared to the Fab-based (IL-15+IL-21) format, the scFv-based (IL-15+IL-21) format displayed a tendency towards higher activity in targeted and lower activity in untargeted settings, emphasizing the targeted concept. Thus, this study underscores the importance of molecular design in developing trifunctional immunocytokines and identified the scFv-based trifunctional (IL-15+IL-21) fusion protein,with the antibody in the central position, as a particularly promising candidate for further drug development.
Keywords: Trifunctional, antibody-cytokine fusion proteins, common gamma chain receptor family, IL-15, IL-7, IL-21, cancer immunotherapy
Received: 19 Sep 2024; Accepted: 01 Apr 2025.
Copyright: © 2025 Möller, Vettermann, Baumann, Pütter and Müller. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Dafne Müller, University of Stuttgart, Stuttgart, Germany
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