Abstract B065: Reprogramming the Tumor Microenvironment Enables T Cell-Mediated Immunotherapies in Pediatric Solid Cancers

Abstract T cell-mediated immunotherapies are extensively investigated across a range of malignancies, demonstrating dramatic therapeutic response in certain entities. In contrast, clinical activity in pediatric cancers thus far is limited. Particularly cancer entities, characterized by an immune excluded or immune deserted tumor microenvironment (TME), also referred to as immunologically “cold”, a prevalent phenotype in pediatric solid cancers, are less accessible for T cell-mediated immunotherapies. Reprogramming the TME towards an inflammatory immune phenotype represents a novel and potentially highly efficient therapeutic strategy to overcome this obstacle and significantly improve the efficacy of T cell-mediated immunotherapies. In this study, we first screened a comprehensive multi-omics data set of relapsed pediatric cancers on signatures and targets associated with an immunologically “cold” TME, characterized among others by low T cell infiltrates, low INFg-signature and abundance of M2-like polarized macrophages. We identified high B7-H3 expression as a functionally linked target structure. To reprogram the TME of pediatric solid cancers by interleukin (IL) 2 and IL12 signals, we developed a novel fusion protein consisting of B7-H3 binding, as well as IL2-receptor and IL12-receptor agonistic domains. This molecule allows targeted delivery and accumulation of the immunostimulatory payload in the TME, limiting toxicities observed after IL2 and IL12 therapies. In vitro, the fusion protein induced NK activation, IFNγ secretion, and M2-to-M1 macrophage polarization. For in vivo evaluation, we utilized orthotopic xenograft and a fully immune competent syngeneic mouse model of rhabdomyosarcoma, resistant to chimeric antigen receptor (CAR) T cell therapy. In the xenograft model, systemic application of the fusion protein prior to CAR-T application results in complete eradication of established tumors. Functional analyses by high-content imaging, reveal in situ TME-reprogramming characterized by M2-like to M1-like macrophage conversion and significant infiltration of functionally active T and CAR-T cells. Strikingly, systemic application of the fusion protein as a single agent therapy is sufficient to induce sustained immunological clearance of established tumors in immunocompetent mice with limited toxicities. Detailed cellular and molecular mechanisms are studied by longitudinal single nuclear RNA sequencing and high content imaging. Here, we demonstrate a novel class of TME reprogramming fusion proteins, efficiently delivering an immunostimulatory payload in the TME of (pediatric) solid cancer after systemic application. By inducing an inflammatory cascade, these compounds “heat up” immunologically “cold” tumors, enabling T cell infiltration and antitumoral activity. Our findings have important clinical implications possibly broadening the group of cancer patients benefitting from T cell-mediated immunotherapies. Clinical translation of the compound is ongoing with a phase I/II trial in preparation. Citation Format: Simon Krost, Moustafa Moustafa-Oglou, Marta Emperador Melero, Roland Imle, Tom Fischer, Anke King, Lennart Schlager, Sophia Scheuermann, Daniel Blösel, Robert Autry, Stefan M. Pfister, Olaf Witt, Kristian Pajtler, Ana Banito, Karin Schilbach, Stephen D. Gillies, Christian M. Seitz. Reprogramming the Tumor Microenvironment Enables T Cell-Mediated Immunotherapies in Pediatric Solid Cancers [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B065.