Fig 1: hBTE significantly extends the survival of patient‐derived xenograft models of primary and recurrent GBM. (A–C) Kaplan–Meier survival analyses of mice bearing primary GBM PDX tumors treated with hBTE, NC‐BTE, or saline. MS, median survival. (A) GBM6: hBTE (n = 10), NC‐BTE (n = 12), saline (n = 8). (B) GBM12: hBTE (n = 11), saline (n = 10). (C) GBM38: hBTE (n = 10), saline (n = 10). (D) Bulk RNA‐seq of matched GBM samples (GSE4271) comparing IL13RA2 between primary and recurrent tumors. Paired Wilcoxon signed‐rank test, one‐sided (Primary > Recurrent). n = 23. (E) Paired dot plot and summary table by patient with histological scoring of matched GBM samples showing comparable IL13RA2 protein expression in primary and recurrent tumors. Paired Wilcoxon signed‐rank test, one‐sided (Primary > Recurrent). n = 18. (F–I) Kaplan–Meier survival analyses and histological evaluation of mice bearing therapy‐resistant GBM PDX tumors treated with hBTE, NC‐BTE, or saline. (F) GBM6r model: hBTE (n = 9), NC‐BTE (n = 7), saline (n = 7). (G) H&E staining of brains from long‐term surviving GBM6r‐bearing mice treated with hBTE. (H) GBM12r model: hBTE (n = 12), saline (n = 11). (I) H&E staining of brains from long‐term surviving GBM12r‐bearing mice treated with hBTE. Survival statistical significance of Kaplan–Meier survival analyses was determined by the log‐rank test relative to saline controls.
Fig 2: Humanized IL13RA2xCD3 bispecific T cell engager shows potent and antigen‐dependent activity against GBM cells. (A) AlphaFold‐predicted structure and schematic of humanized IL13RA2xCD3 bispecific T cell engager (hBTE), composed of anti‐IL13RA2 scFv, CD3E‐targeting scFv, and human Fc domain. (B) ELISA binding analysis of three hBTE scFv configurations to IL13RA2. (C) Chromium‐51 release assay showing dose‐dependent hBTE‐induced T cell–mediated killing of three hBTE scFv configurations, with hBTEv1 showing the strongest binding. (D) Flow cytometry mean fluorescent intensity showing dose‐dependent hBTE binding to CD4 and CD8 T cells using AF647‐conjugated anti‐His‐tag antibody. (E) Flow cytometric histogram of IL13RA2 surface expression on GBM6, GBM12, GBM39, and GBM39 PDX cell lines. (F) hBTE binding at varying concentrations to GBM6 and GBM39, assessed by flow cytometry and presented as mean fluorescent intensities. (G) Chromium‐51 release assay showing dose‐dependent hBTE‐induced T cell–mediated killing of GBM6, GBM12, and GBM38, but no effect on GBM39. (H) hBTE induced cytokine release in GBM6 co‐cultures with T cells from both healthy donors and GBM patients, whereas little to no cytokine production was observed in GBM39 co‐cultures, demonstrating antigen‐dependent activity.
Fig 3: hBTE enhances T cell infiltration and improves outcomes in models of brain metastases and subcutaneous extracranial tumors. (A) IL13RA2 mRNA expression across 25 tumor types in the TCGA Pan‐Cancer dataset, showing variable, patient‐specific expression. (B) Chromium release assay showing dose‐dependent hBTE‐induced T cell–mediated killing of MDA‐MB‐231Br cells treated with or without XRT. Two‐way ANOVA (factors: XRT‐treatment and hBTE Concentration); p‐value corresponds to the main effect of hBTE Concentration. (C) Survival curves of nude mice bearing intracranial MDA‐MB‐231Br tumors treated with hBTE (50 µg, i.v., twice weekly), radiotherapy (XRT; 5 × 2 Gy), or both. MS, median survival. Statistical significance was determined by the log‐rank test relative to the saline control. (D) Representative immunofluorescent images of brain sections from control and XRT‐treated mice following hBTE administration, showing hBTE signal distribution. Blue, DAPI; red, tumor; green, hBTE. Scale bar, 375 µm. (E) Quantification of hBTE fluorescence intensity in XRT‐treated mice compared to controls. Statistical significance was determined by Student's t‐test. (F) Chromium release assay showing dose‐dependent hBTE‐mediated killing of NCI‐H460 lung cancer cells. (G) Schematic of the in vivo experimental design: human PBMCs were adoptively transferred into NSG MHC I/II DKO mice, followed by subcutaneous implantation of NCI‐H460 tumors and hBTE treatment. (H) Quantification of tumor burden in mice treated with hBTE (n = 8) or saline (n = 7) was measured as tumor weight and volume. Unpaired Welch's t‐test. (I) Histological analysis of tumors showing significantly increased tumor cell death in hBTE‐treated mice, assessed by viable‐to‐necrotic tissue ratio. (J) Quantification of intratumoral CD3+ T cells, showing significantly increased infiltration in hBTE‐treated tumors. Unpaired Welch's t‐test.
Fig 4: IL13RA2 is highly expressed in malignant gliomas. (A) IL13RA2 mRNA expression across 24 tissue types in the Adult Genotype Tissue Expression (GTEx) Project, showing low expression in all tissues except Testis. (B) IL13RA2 mRNA expression in the TCGA‐GBM/LGG dataset stratified by glioma genotype. A, IDH‐mutant Astrocytoma (n = 258); ODG, IDH‐mutant oligodendroglioma (n = 169); GBM, IDH‐WT Glioblastoma (n = 229). (C) Kaplan–Meier curves of overall survival in the TCGA‐GBM dataset based on IL13RA2 expression levels. High (Q3; n = 59) and low (Q1; n = 59) expression groups were defined using quantile cutoffs. Log‐rank test. (D) UMAP visualization of single‐cell RNA sequencing data from 110 GBM patients showing IL13RA2 expression predominantly restricted to malignant cells. (E) Multiplex sequential immunofluorescence (seqIF) of three GBM patient samples showing varying IL13RA2 protein expression. Scale bar, low magnification: 800 µm; high magnification: 200 µm.
Fig 5: Humanization of anti‐IL13RA2 antibody. (A) Schematic of the humanization strategy. Murine CDRs were grafted onto four human VH and four human VL frameworks, generating a panel of 16 fully humanized antibody variants. (B) Western blot of selected humanized antibodies under reducing and non‐reducing conditions, showing high purity, correct assembly, and expected molecular weights. (C) Biacore T200 sensorgrams showing binding kinetics of selected humanized antibodies and the original chimeric antibody to human IL13RA2. The table summarizes kinetic parameters. (D) ELISA measuring binding of humanized VH1+VL1 antibody and chimeric antibody to human IL13RA2. EC50 values indicate comparable binding. UD, undetectable.
Supplier Page from R&D Systems, a Bio-Techne Brand for IL-13 R alpha 2 Fc Chimera Protein
Available conjugates: Sizes Available: 100 ug