Synergistic microglial activation and tumor cell killing for improved GBM response

项目摘要/摘要 胶质母细胞瘤(GBM)是成人最常见、最具侵袭性的原发脑肿瘤。目前的治疗方法仍然存在 在提高总体存活率方面不成功；因此，确定治疗基底膜的新疗法是至关重要的。我们有 开创了一种以免疫毒素(IT)为基础的新型细胞毒疗法，D2C7-IT，以表皮生长为目标 因子受体(EGFR)和突变型EGFR变异体III(EGFRvIII)是GBM的致癌驱动基因。在……里面 临床前研究，D2C7-IT靶向并杀死大量肿瘤细胞，延长生存时间，但 无法在所有治疗的动物中产生治愈，因为存在高度免疫抑制的GBM 微环境。基底膜微环境中的大多数免疫细胞与肿瘤相关。 巨噬细胞(TAMs)，促进肿瘤细胞生长，抑制抗肿瘤T细胞反应。因此， 消除介导的免疫抑制有望增强D2C7-IT诱导的抗肿瘤免疫 回应。CD40是一种免疫共刺激分子，它的激活被认为可以重新教育TAM，以及 也能诱导T细胞反应。因此，推动本提案的中心假设是克服 通过CD40共刺激的免疫抑制和促肿瘤活性将改善 细胞毒D2C7-IT疗法的疗效。因此，我们的初步研究表明：(1)在 小鼠脑胶质瘤模型D2C7-IT+αCD40协同作用延长生存期 治疗，(2)脑内驻留的小胶质细胞是主要的抗原提呈细胞(APC)被联合激活 治疗，以及(3)CD8CD40T细胞只有在联合使用时才能发挥抗肿瘤作用 用细胞毒D2C7-IT。我们的结果有力地表明αCD40改变了TAMs的发育或活性 并激活小胶质细胞/T细胞。D2C7-IT联合αCD_(40)治疗人肝癌的实验研究 相关的脑肿瘤模型及其作用机制的深入研究将对临床有很大的帮助。 D2C7-IT+αCD40疗法的翻译。因此，我们建议追求三个具体目标来表征 D2C7-IT+αCD40抗肿瘤疗效、再教育及小胶质细胞/T细胞活化机制：目的1： 评价αCD40能否克服的免疫抑制并增强D2C7-IT的疗效 免疫活性胶质瘤模型。目的2：确定小胶质细胞CD40/MHCII分子是否为介体 D2C7-IT+αCD40抗肿瘤免疫应答。目的3：确定D2C7-IT+αCD40治疗是否有刺激作用 CD8+效应T细胞反应能够消除抗原阳性和抗原阴性的肿瘤。这个 拟议的研究具有重要意义，因为它将导致开发一种治疗策略 同时杀伤肿瘤细胞，逆转免疫抑制，激活小胶质细胞和T细胞，以及 最终可以在临床上进行翻译和测试。

PROJECT SUMMARY/ABSTRACT Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Current therapies remain unsuccessful in improving overall survival; thus, the identification of novel therapies for GBM is critical. We have pioneered a novel, targeted immunotoxin (IT)-based cytotoxic therapy, D2C7-IT, that targets epidermal growth factor receptor (EGFR) and mutant EGFR variant III (EGFRvIII), established driver oncogenes of GBM. In preclinical studies, D2C7-IT targets and kills a substantial number of tumor cells and prolongs survival but is unable to generate cures in all treated animals because of the presence of a highly immunosuppressive GBM microenvironment. The majority of the immune cells in the GBM microenvironment are tumor-associated macrophages (TAMs), which promote tumor cell growth and inhibit antitumor T cell responses. Therefore, eliminating TAM-mediated immunosuppression is anticipated to enhance D2C7-IT-induced antitumor immune responses. CD40 is an immune co-stimulatory molecule whose activation is known to re-educate TAMs, and also induce T cell responses. Thus, the central hypothesis driving the present proposal is that overcoming TAM immunosuppression and tumor-promoting activities via CD40 co-stimulation will improve the efficacy of the cytotoxic D2C7-IT therapy. Accordingly, our preliminary studies have demonstrated that (1) in a mouse glioma model, D2C7-IT+αCD40 functions synergistically to prolong survival and generate significant cures, (2) brain resident microglia is the principal antigen-presenting cells (APCs) activated by the combination therapy, and (3) αCD40 treatment engages CD8+ effector T cells that are antitumorigenic only when combined with cytotoxic D2C7-IT. Our results strongly imply that αCD40 alters either the development or activity of TAMs in GBM and activates microglia/T cells. Demonstrating the antitumor efficacy of the D2C7-IT+αCD40 therapy in relevant brain tumor models and gaining insights into their mechanism of action will greatly aid in the clinical translation of D2C7-IT+αCD40 therapy. Therefore, we propose to pursue three Specific Aims to characterize D2C7-IT+αCD40 antitumor efficacy, TAM re-education, and microglia/T cell activation mechanisms: Aim 1: Evaluate whether αCD40 overcomes TAM immunosuppression and enhances D2C7-IT efficacy in two preclinical immunocompetent glioma models. Aim 2: Define whether microglial CD40/MHCII molecules are the mediators of D2C7-IT+αCD40 antitumor immune response. Aim 3: Determine whether D2C7-IT+αCD40 therapy stimulates CD8+ effector T cell response capable of eliminating antigen-positive as well as antigen-negative tumors. The proposed research is significant because it will result in the development of a therapeutic strategy for simultaneous tumor cell killing, reversal of TAM immunosuppression, activation of microglia and T cells, and ultimately could be translated and tested in the clinic.