Abstract A036: The recombinant immunotoxin GB13 demonstrates significant therapeutic potential for H3.3K27M mutant DIPG with its efficacy linked to the activation of Golgi-to-ER retrograde transport pathways

Abstract GB13 is a recombinant immunotoxin composed of a truncated form of Pseudomonas exotoxin A and an engineered IL-13 mutein, E13K. Its mechanism of action is initiated by binding to the IL-13 receptor alpha 2 (IL13Rα2), which is frequently overexpressed on the surface of various cancer cells. This binding facilitates the internalization of the immunotoxin. Once inside the cell, the active domain of the toxin is released into the cytoplasm, where it catalyzes the ADP-ribosylation of eukaryotic elongation factor 2 (eEF-2). This action effectively halts de novo protein synthesis and induces apoptosis, or programmed cell death. Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and difficult-to-treat pediatric brain tumor characterized by the significant upregulation of IL13Rα2. The overexpression of this receptor makes GB13 a promising therapeutic agent for DIPG. The potential of GB13 is further underscored by the established link between the histone 3.3 (H3.3) K27M mutation, a hallmark of DIPG, and the increased expression of IL13Rα2. The presence of the H3.3 K27M mutation provides a strong rationale for utilizing GB13 in this patient population. In vitro drug dose-response assays using patient-derived DIPG cell lines confirmed the therapeutic potential of GB13. The H3.3K27M mutant cell lines, PBT22 and PBT29, demonstrated high sensitivity to GB13, with IC50 values of 9.318 ng/ml and 0.9834 ng/ml, respectively In contrast, the H3.3 wild-type cell line, PBT24, was resistant to GB13, exhibiting IC50 values of 392.7 ng/ml. To further investigate its efficacy, the cytotoxic activity of GB13 was tested across a panel of adult glioblastoma (GBM) patient-derived xenograft (PDX) models. Gene set enrichment analysis (GSEA) of transcriptomic data from these PDX models revealed a correlation between sensitivity to GB13 and the activation of pathways involved in Golgi-to-ER retrograde transport. This requirement for retrograde transport aligns with the known intracellular trafficking route of GB13, which necessitates such transport to the endoplasmic reticulum to execute its cytotoxic function. Single-sample GSEA (ssGSEA) scores were calculated for Golgi-to-ER retrograde transport-related pathways within the TCGA-GBM transcriptome dataset. Notably, these ssGSEA scores show a negative correlation with patient age, suggesting that the efficacy of GB13 may be dependent on tumor subtypes and demographic factors. Furthermore, ssGSEA was performed for four DIPG cell lines: PBT22, PBT29, SU-DIPG-IV, and SU-DIPG-XVII. PBT29, an H3.3K27M mutant, was the most sensitive to GB13 and exhibited the highest enrichment scores in Golgi-to-ER retrograde transport-related pathways. In conclusion, the potent and selective activity of GB13 in H3.3K27M mutant DIPG cells, combined with the identification of Golgi-to-ER retrograde transport pathways as associated predictive biomarkers, positions GB13 as a highly promising therapeutic candidate for this devastating disease. Citation Format: Yue Hao, Nanyun Tang, Valerie De Luca, Michael E. Berens. The recombinant immunotoxin GB13 demonstrates significant therapeutic potential for H3.3K27M mutant DIPG with its efficacy linked to the activation of Golgi-to-ER retrograde transport pathways [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr A036.