Combinatorial Immunotherapy using a Multivalent Drug Conjugate for GBM Treatment

总结 胶质母细胞瘤（GBM）的治疗代表了医学上未满足的需求。我们一直在寻找 使用持续进化的细胞毒素递送有效的靶向和特异性细胞毒素的治疗方法 对流增强输送。GBM患者过度表达白细胞介素13受体α 2（IL-13RA 2）， EphA2、EphA3和EphB2受体存在于GBM的各种病理生理区室中 并且所有四种都在肿瘤核心的肿瘤细胞和局部浸润的肿瘤细胞中表达，而EphA2 也存在于肿瘤新生血管中。此外，IL-13 RA 2、EphA2和EphA3与IL-13 RA 2、EphA2和EphA3相关，并发挥作用。 神经胶质瘤干细胞样细胞的病理生物学中的关键作用。最后，在M2 GBM中发现EphA3受体。 相关巨噬细胞。因此，总的来说，IL-13RA 2、EphA2、EphA3和EphB2在大肠杆菌中过表达。 主要GBM区室显示参与肿瘤进展和/或对治疗的抗性。中 第一种方法，我们在患有自发性胶质瘤的狗中进行了I期临床试验，这代表了 使用靶向IL-13RA 2和EphA2受体的细胞毒素的混合物的人类疾病的忠实模型。我们 观察到异常的抗肿瘤反应，包括几次几乎完全消退， 生存和良好的生活质量在这个剂量探索试验，在没有毒性。此外，我们还发现了 免疫系统在治疗过程中在这些结果的鼓舞下，我们提出了一个新的想法， 用一种药物化合物靶向所有四种受体而不是两种受体。Eph受体配体之一， ephrinA5（eA5）结合EphA2、EphA3和EphB2受体。因此，我们生成了一个基于eA5的代理 和使用IgG1支架靶向所有四种受体的IL-13突变体（QUAD）。在我们最初的实验中， QUAD与多柔比星（Dox）的衍生物或假单胞菌外毒素A的衍生物缀合， PE38QQR，以产生单一药剂，并且这些药物缀合物保持其结合 对靶向受体的亲和力，同时对GBM细胞表现出显著的杀伤活性。QUAD- Dox和QUAD-PE38QQR缀合物已经在狗中显示出显著的、持久的抗肿瘤作用 无毒性的自发性神经胶质瘤：治疗犬的肿瘤体积消退率分别为60%、88%和91%， 分别最近，我们将QUAD与微管破坏剂DM1结合。QUAD-DM1是 对GBM细胞极其有效，IC 50在低飞摩尔范围内，比Dox/PE缀合物好约50倍。 因此，我们将通过以下具体目标继续这一令人兴奋的研究路线。在具体目标1中， 将用QUAD-DM1治疗患有自发性新诊断和复发性高级别胶质瘤的犬。在 具体目标2，我们将检查免疫反应和复发肿瘤的表型和基因型， QUAD-DM1治疗。我们的方法解决了肿瘤间和肿瘤内异质性的关键问题 并引起原位接种或所谓的"肿瘤发炎"效应。我们设想这次全面进攻 被我们称为“分子切除术”，将导致更有效的管理GBM。

SUMMARY Treatment of glioblastoma (GBM) represents an unmet need in medicine. We have been pursuing a therapeutic approach of delivering potent targeted and specific cytotoxins using continuously evolving convection-enhanced delivery. Patients with GBM over-express interleukin 13 receptor alpha 2 (IL-13RA2), EphA2, EphA3 and EphB2 receptors that are present in various pathophysiological compartments of GBM and all four are expressed in tumor cells of the core of tumor, and in locally-infiltrating tumor cells, while EphA2 is also found in tumor neovasculature. Further, IL-13RA2, EphA2, and EphA3 are associated with, and play crucial roles in, the pathobiology of glioma stem-like cells. Finally, the EphA3 receptor are found in M2 GBM- associated macrophages. Thus, collectively, IL-13RA2, EphA2, EphA3 and EphB2 are over-expressed in principal GBM compartments shown to be involved in tumor progression and/or resistance to therapies. In a first-of-kind approach, we performed Phase I clinical trial in dogs with spontaneous gliomas, which represents a faithful model of human disease, using a cocktail of cytotoxins targeting IL-13RA2 and EphA2 receptor. We observed exceptional anti-tumor responses, including several near complete regressions, prolongation of survival and excellent quality of life in this dose-finding trial, at no toxicity. In addition, we found evidence for immune system activation during the therapy. Encouraged by these results, we pursued the novel idea of targeting all four receptors instead of two with one pharmaceutical compound. One of the Eph receptor ligands, ephrinA5 (eA5), binds EphA2, EphA3 and EphB2 receptors. We have thus generated an agent based on eA5 and IL-13 mutants targeting all four receptors using an IgG1 scaffold (QUAD). In our initial experiments, the QUAD was conjugated to derivatives of Doxorubicin (Dox) or a derivative of Pseudomonas exotoxin A, PE38QQR, to generate single pharmaceutical agents and these drug conjugates retained their binding affinities towards the targeted receptors while demonstrating prominent killing activity on GBM cells. QUAD- Dox and QUAD-PE38QQR conjugates have already shown prominent, long-lasting anti-tumor effects in dogs with spontaneous glioma at no toxicity: 60, 88, and 91% of tumor volume regression in the treated dogs, respectively. Recently, we have conjugated QUAD to DM1, a microtubule-disrupting agent. The QUAD-DM1 is extremely potent on GBM cells with IC50s in low femtomolar range, ~50x better than the Dox/PE conjugates. Therefore, we will continue this exciting line of research through Specific Aims as follows. In Specific Aim 1, we will treat dogs with spontaneous newly diagnosed and recurrent high-grade gliomas with QUAD-DM1. In Specific Aim 2, we will examine immune responses and the phenotype and genotype of recurring tumors in the course of QUAD-DM1 therapy. Our approach addresses crucial issues of inter- and intra-tumoral heterogeneity and evokes an in situ vaccination or so called “tumor inflaming” effect. We envision that this all-out assault, termed by us “molecular resection”, will result in a more effective management of GBM.