Neural Stem Cell Carriers for Glioblastoma Immunotherapy

胶质母细胞瘤(GBM)是成人最具侵袭性的恶性脑癌。被诊断出患有 GBM的治疗选择有限，生存期短。现有的一个主要问题 治疗方法是缺乏对肿瘤细胞的特异性，这导致了实质性的治疗。 毒性。抗体介导的肿瘤相关抗原的特异性靶向已经成为一种成功的策略 癌症治疗，因为它限制了全身输注药物的非靶点效应。这样的基因修改 抗体结合有效的递送策略可以极大地提高这些药物的抗肿瘤效果 分子。一种这样的修饰是双特异性串联单链抗体(BiscFv)，它能促进T细胞- 肿瘤细胞相互作用，进而杀死肿瘤细胞。然而，BiscFv的半衰期很短，而且很快 清除，需要频繁或持续输液才能达到治疗效果。我们建议 通过产生产生BiscFv的神经干细胞(NSCs)来克服这些障碍。NSC能够 在全身、局部和鼻腔给药后追踪脑瘤细胞，并有效地提供治疗 GBM临床前模型中肿瘤部位的有效载荷。分泌BiscFv的神经干细胞可以直接与 自体患者的T细胞用于产生局部免疫反应，旨在根除肿瘤。 最近，我们开发并鉴定了一种针对细胞表面IL13R2的单抗 在胶质瘤细胞中选择性表达的受体，但不能在正常脑细胞或其他组织中表达。我们 证明了工程化单链抗体具有独特的特异性和高亲和力。 IL13R2，并成功地将工程化腺病毒和治疗性CAR T细胞重定向至IL13R2- 胶质瘤细胞在临床前胶质瘤模型中的表达。除了被过度表达在 大多数GBM、IL13R2的表达与高度侵袭性的间充质细胞有关 亚型基因表达特征和较差的患者预后，所有这些都表明靶向IL13R2- 表达胶质瘤细胞可以改善GBM患者的预后。我们假设NSCs被设计成 分泌双特异性IL13Rα2xCD3单链抗体可促进抗肿瘤免疫 通过T细胞与GBM细胞的激活和接触来应答。推进这项治疗 临床应用将通过R21阶段完成，在此期间我们将重点关注详细的 用于生产功能性双链抗体IL13Rα2xCD3的双链抗体神经干细胞的分析和鉴定及其表达能力 激活T细胞并在体外诱导对表达IL13Rα2的胶质瘤细胞的杀伤作用。在R33阶段， 我们将使用免疫活性和患者来源的方法在体内评估BiscFvNSCs的功能反应 骨髓间充质干细胞异种移植模型。这将包括BiscFvNSC本地生产BiscFv IL13Rα2xCD3的能力， 结合T细胞和胶质瘤细胞，并诱导出强大的抗胶质瘤活性。在每个阶段，我们都将实现 量化里程碑，这将进一步确保BiscFvNSCs作为一种新的治疗方式的优化 用于GBM治疗。

Glioblastoma (GBM) is the most aggressive malignant brain cancer in adults. People diagnosed with GBM have limited therapeutic options and short survival expectancies. A major problem with existing therapeutic approaches is their lack of specificity for neoplastic cells, which results in substantial treatment toxicity. Antibody-mediated specific targeting of tumor-associated antigens has been a successful strategy for cancer therapy as it limits the off-target effect of systemically infused drugs. Genetic modifications of such antibodies coupled with efficient delivery strategies can greatly improve the anti-tumor efficacy of these molecules. One such modification is bi-specific tandem single–chain antibodies (biscFv) that promote T-cell- tumor cell interactions that, in turn, kill the tumor cells. However, biscFv have short half-lives and fast clearance, necessitating frequent or continuous infusions to achieve therapeutic effect. We propose to overcome these hurdles through the generation of neural stem cells (NSCs) producing biscFv. NSCs are able to track brain tumor cells after systemic, local, and intranasal delivery, and efficiently deliver therapeutic payload to tumors sites in preclinical models of GBM. NSCs secreting biscFv can be directly mixed with autologous patients T cells for the production of a local immune response aimed at eradicating tumors. Recently, we developed and characterized a monoclonal antibody specifically targeting IL13R2, a cell surface receptor that is selectively expressed in glioma cells, but not normal brain cells or other tissues. We demonstrated that engineered single-chain antibody retains an exclusive specificity as well as a high affinity to IL13R2, and successfully re-targets engineered adenovirus and therapeutic CAR T cells to IL13R2- expressing glioma cells in pre-clinical models of GBM, in vitro and in vivo. In addition to being overexpressed in the majority of GBMs, IL13R2 expression has been associated with the highly aggressive mesenchymal subtype gene expression signature and poorer patient prognosis, all of which suggest that targeting IL13R2- expressing glioma cells could improve GBM patient outcomes. We hypothesize that NSCs engineered to secrete bi-specific tandem IL13Rα2xCD3 scFv antibody (biscFvNSCs) will promote anti-tumor immune response through the activation and engagement of T cells with GBM cells. Advancing this therapeutic for clinical application will be accomplished through R21 phase, during which we will focus on the detailed analysis and characterization of biscFvNSCs for production of functional biscFv IL13Rα2xCD3 and the ability to activate T cells and elicit cytotoxic effect against IL13Rα2-expressing glioma cells in vitro. During R33 phase, we will evaluate functional responses of biscFvNSCs in vivo, using immune-competent and patient-derived xenograft models of GBM. This will include the ability of biscFvNSCs to locally produce biscFv IL13Rα2xCD3, engage T and glioma cells, and elicit potent anti-glioma activity. During each phase, we will achieve quantitative milestones, which will further ensure the optimization of biscFvNSCs as a new therapeutic modality for GBM treatment.