Neural Stem Cell Carriers for Glioblastoma Immunotherapy

胶质母细胞瘤（GBM）是成人中最具侵袭性的恶性脑癌。人被确诊为 GBM的治疗选择有限，生存期短。现有的一个主要问题是 治疗方法缺乏对肿瘤细胞的特异性，这导致实质性治疗 毒性抗体介导的肿瘤相关抗原的特异性靶向已经成为治疗肿瘤的成功策略。 癌症治疗，因为它限制了全身输注药物的脱靶效应。基因改造， 抗体与有效的递送策略偶联可以大大提高这些药物的抗肿瘤功效。 分子。一种这样的修饰是双特异性串联单链抗体（biscFv），其促进T细胞免疫应答。 肿瘤细胞相互作用，反过来杀死肿瘤细胞。然而，biscFv具有短的半衰期和快速的 清除，需要频繁或连续输注以达到治疗效果。我们建议 通过产生产生biscFv的神经干细胞（NSC）克服这些障碍。NSC能够 在全身、局部和鼻内递送后追踪脑肿瘤细胞， 在GBM的临床前模型中有效载荷至肿瘤部位。分泌biscFv的NSC可以直接与 自体患者T细胞用于产生旨在根除肿瘤的局部免疫应答。 最近，我们开发并鉴定了特异性靶向IL 13 R β 2的单克隆抗体， 在神经胶质瘤细胞中选择性表达，但在正常脑细胞或其他组织中不表达。我们 证明了工程化单链抗体保留了排他性的特异性以及高亲和力， IL 13 R β 2，并成功地将工程化的腺病毒和治疗性CAR T细胞重新靶向IL 13 R β 2。 在GBM的临床前模型中，在体外和体内表达胶质瘤细胞。除了过度表达外， 大多数GBM，IL 13 R β 2表达与高度侵袭性间质细胞浸润有关。 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IL 13 R β 2- 表达胶质瘤细胞可以改善GBM患者的结果。我们假设，神经干细胞工程， 分泌双特异性串联IL 13 R α 2xCD 3 scFv抗体（biscFvNSCs）将促进抗肿瘤免疫 通过T细胞与GBM细胞的活化和接合来产生免疫应答。推进这种治疗 临床应用将通过R21阶段完成，在此期间，我们将专注于详细的 用于产生功能性biscFv IL 13 Ra 2xCD 3的biscFvNSC的分析和表征以及 体外激活T细胞并引发针对表达IL 13 R α2的胶质瘤细胞的细胞毒效应。在R33阶段， 我们将使用免疫活性的和患者来源的细胞因子， GBM的异种移植模型。这将包括biscFvNSC局部产生biscFv IL 13 Ra 2xCD 3的能力， 接合T细胞和神经胶质瘤细胞，并引发有效的抗神经胶质瘤活性。在每个阶段，我们将实现 定量里程碑，这将进一步确保biscFvNSC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.