D2C7免疫毒素（IT）是一种双特异性IT，靶向野生型表皮生长因子受体（EGFR）和突变型EGFR III（EGFRvIII）蛋白。在少数脑胶质母细胞瘤患者中，D2C7-IT表现出令人鼓舞的生存疗效。我们假设免疫抑制是导致D2C7-IT疗效受限的原因。为了改善反应率并抑制免疫抑制作用，我们将D2C7-IT肿瘤细胞杀伤与αCD40抗原递呈细胞共刺激相结合。在小鼠胶质瘤模型中，D2C7-IT+αCD40治疗通过激活巨噬细胞和微胶质细胞的促炎性表型、促进长期肿瘤特异性CD8+ T细胞免疫、实现了治愈效果。D2C7-IT+αCD40治疗增加了肿瘤内部的Slamf6+CD8+ T细胞，降低了终末耗竭的CD8+ T细胞。D2C7-IT+αCD40治疗刺激了肿瘤内部的CD8+ T细胞增殖，并在荷瘤小鼠中实现了治愈效果，尽管给小鼠注射FTY720导致其外周T细胞被隔离。肿瘤转录组分析确定了CD40上调、模式识别受体、细胞衰老和免疫应答途径的激活是D2C7-IT+αCD40抗肿瘤反应的驱动因素。为了确定潜在的应用，免疫组织化学染色证实了人类脑胶质母细胞瘤组织切片中CD40表达的存在。这些有希望的临床前数据使我们开始了一项D2C7-IT+αhCD40治疗恶性脑胶质母细胞瘤患者的1期研究（NCT04547777），以进一步评估该治疗在人体中的作用。

D2C7-immunotoxin (IT), a dual-specific IT targeting wild-type epidermal growth factor receptor (EGFR) and mutant EGFR variant III (EGFRvIII) proteins, demonstrates encouraging survival outcomes in a subset of patients with glioblastoma. We hypothesized that immunosuppression in glioblastoma limits D2C7-IT efficacy. To improve the response rate and reverse immunosuppression, we combined D2C7-IT tumor cell killing with αCD40 costimulation of antigen-presenting cells. In murine glioma models, a single intratumoral injection of D2C7-IT+αCD40 treatment activated a proinflammatory phenotype in microglia and macrophages, promoted long-term tumor-specific CD8+ T cell immunity, and generated cures. D2C7-IT+αCD40 treatment increased intratumoral Slamf6+CD8+ T cells with a progenitor phenotype and decreased terminally exhausted CD8+ T cells. D2C7-IT+αCD40 treatment stimulated intratumoral CD8+ T cell proliferation and generated cures in glioma-bearing mice despite FTY720-induced peripheral T cell sequestration. Tumor transcriptome profiling established CD40 up-regulation, pattern recognition receptor, cell senescence, and immune response pathway activation as the drivers of D2C7-IT+αCD40 antitumor responses. To determine potential translation, immunohistochemistry staining confirmed CD40 expression in human GBM tissue sections. These promising preclinical data allowed us to initiate a phase 1 study with D2C7-IT+αhCD40 in patients with malignant glioma (NCT04547777) to further evaluate this treatment in humans.