Sub-component for Institution # 16-01848 Novel moleculary targeted therapy of GBM

胶质母细胞瘤（GBM）患者预后不佳。我们发现超过90%的GBM患者过度表达正常脑中缺失的白细胞介素13受体α 2 （IL-13RA2）和EphA2受体。最近，我们已经证明EphA3受体在GBM中也过表达，并且其表达模式与EphA2不同。IL-13RA2、EphA2和EphA3存在于GBM肿瘤的各个腔室中。这三种受体均在肿瘤核心的肿瘤细胞和局部浸润性肿瘤细胞中表达，EphA2在肿瘤新生血管中也过表达。此外，IL-13RA2、EphA2和EphA3与胶质瘤干细胞（glioma stem-样细胞，GSC）的病理生物学相关，并在GSC中发挥关键作用。IL-13RA2参与GSC特性，EphA2和EphA3共同驱动GSC的自我更新和致瘤性。最后，EphA3受体可以在单核细胞来源的gbm浸润细胞，肿瘤相关巨噬细胞（TAM）中很容易检测到。因此，总的来说，IL-13RA2、EphA2和EphA3在主要的GBM区室中表达，表明与肿瘤进展和/或治疗耐药性有关。Eph受体配体之一ephrinA5 （eA5）结合EphA2和EphA3，也独特地结合EphB2受体；后者也在GBM细胞上特异性表达。在目前的项目中，我们将追求用一种药物化合物靶向所有四种受体的新思路。该双价化合物可识别IL- 13RA2、EphA2、EphA3和EphB2，并向GBM肿瘤传递药物，特异性杀伤肿瘤细胞和肿瘤环境细胞，促进其生长。我们还分离出一种小肽Pep-1L，它特异性识别IL-13RA2，诱导受体内化，并与小鼠颅内肿瘤结合。这些和其他特性使肽成为增加药物/标签进入肿瘤的理想载体。我们将通过两个具体目标继续这一令人兴奋的研究。在第一个目标中，我们将生产一种有效的双价细胞毒素QUAD-CTX，同时靶向IL-13RA2、EphA2、EphA3和EphB2受体。我们将产生一种化学偶联物与改良的化疗药物，可以通过血脑屏障（BBB）自己，命名为WP1244。药物偶联物将在GBM的不同腔室的体外模型和体内进行测试。在第二个目标中，我们将利用与IL-13RA2结合的肽进行有效的靶向全身或局部区域化疗，监测血脑屏障的破坏和对治疗的反应。我们将利用内化肽Pep-1L与WP1244 （Pep-1L- ctx）结合，并在表达IL-13RA2的GBM模型中进行测试，以验证该结合物穿透血脑屏障和/或血脑肿瘤屏障的能力。我们还将生成基于肽的成像探针。我们期望这种对GBM的全面攻击将转化为患者明确的持久反应。

Patients with glioblastoma (GBM) have a dismal prognosis. We found that over 90% of patients with GBM over-express both interleukin 13 receptor alpha 2 (IL-13RA2) and EphA2 receptor that are absent in normal brain. More recently, we have demonstrated that the EphA3 receptor is also over-expressed in GBM, and its pattern of expression differs from that of EphA2. IL-13RA2, EphA2, and EphA3 are present in various compartments of GBM tumors. All three receptors are expressed in tumor cells of the core of tumor and in locally-infiltrating tumor cells, while EphA2 is also over-expressed in tumor neovasculature. Further, IL-13RA2, EphA2, and EphA3 are associated with, and play crucial roles in, the pathobiology of glioma stem-like cells (GSC). IL-13RA2 contributes to GSC properties and EphA2 and EphA3 both drive their self-renewal and tumorigenicity. Finally, the EphA3 receptor can be readily detected in GBM-infiltrating cells of monocytic origin, tumor-associated macrophages (TAM). Thus, collectively, IL-13RA2, EphA2, and EphA3 are expressed in principal GBM compartments shown to be involved in tumor progression and/or resistance to therapies. One of the Eph receptor ligands, ephrinA5 (eA5), binds EphA2 and EphA3 and also uniquely the EphB2 receptor; the latter also is expressed specifically on GBM cells. In the current project, we will pursue the novel idea of targeting all four receptors with one pharmaceutical compound. This bi-valent compound will recognize IL- 13RA2, EphA2, EphA3, and EphB2 and deliver a drug to GBM tumors, specifically killing tumor cells and cells of the tumor environment promoting its growth. We have also isolated a small peptide, Pep-1L, which specifically recognizes IL-13RA2, induces receptor's internalization and it is bound by intracranial tumors in mice. These and other properties make the peptide a desirable vehicle to augment access of drugs/labels to tumors. We will continue this exciting line of research through two Specific Aims. In the first Aim, we will produce a potent bi-valent cytotoxin, QUAD-CTX, simultaneously targeting the IL-13RA2, EphA2, EphA3, and EphB2 receptors. We will generate a chemical conjugate with a modified chemotherapeutic which can pass the blood-brain barrier (BBB) on its own, termed WP1244. The drug conjugate will be tested in in vitro models of various compartments of GBM and in vivo. In the second Aim, we will exploit a peptide binding to IL-13RA2 for effective targeted systemic or loco-regional chemotherapy, for monitoring of the disruption of the BBB and responses to treatment. We will exploit internalized peptide Pep-1L, which homes to intracranial tumors, for conjugation to WP1244 (Pep-1L-CTX) and test it in GBM models expressing IL-13RA2 to verify the conjugate's ability to penetrate the BBB and/or blood-brain tumor barrier. We will also generate imaging probes based on the peptide. We expect that this comprehensive assault on GBM will translate into clear-cut durable responses in patients.