Chimeric Anti-Fibulin-3 Reagents For Targeted Therapy Of Glioblastoma

 描述（由申请人提供）：本项目的目标是开发和验证针对胶质母细胞瘤（GBM）的新型生物试剂，GBM是最常见和最具侵袭性的脑肿瘤之一。GBM在脑组织中具有高度侵袭性，并且它们的分散通过细胞毒性和抗血管生成疗法而加剧，从而在肿瘤复发时导致更差的临床表现。解决GBM生物学的新疗法应该能够破坏侵袭或血管形成并使肿瘤细胞敏感，从而在与常规疗法组合时在多个方面引起肿瘤破坏。本项目将重点研究本课题组鉴定的fibulin-3蛋白，该蛋白由GBM细胞分泌，在GBM中具有独特的定位和机制，但在正常脑中不存在。Fibulin-3是Notch通路的新型激活剂，该蛋白质的遗传靶向破坏肿瘤中的Notch信号传导并降低GBM生长和抗性。我们已经产生了针对纤蛋白-3的功能阻断抗体（“mAb428.2”），并且在该项目中，我们将使用它作为起点来产生和验证抗纤蛋白-3重组试剂家族。在具体目标1中，我们提出验证mAb 428.2针对颅内GBM的安全性和有效性，以将其推向研究新药状态。我们假设抗fibulin-3将减少肿瘤生长和血管形成，而没有显著的外周毒性或局部脱靶效应。我们将按照FDA指南研究mAb428.2的特异性、交叉反应性、生物分布、安全性和抗肿瘤功效，旨在完成IND前验证。在具体目标2中，我们建议开发和验证抗fibulin-3单链免疫毒素作为GBM的细胞毒性剂。我们已经从mAb428.2中产生了一个单链可变区片段（scFv），并假设该片段的较小尺寸将改善对GBM的接近性和毒性。我们将结合的scFv与假单胞菌外毒素，并将验证这种新的免疫毒素的特异性，安全性和有效性对颅内GBM。最后，使用纤蛋白-3 scFv，在特定目标3中，我们提出产生并测试针对纤蛋白-3的嵌合抗原受体（CAR）。我们假设在T淋巴细胞中表达的纤蛋白-3结合CAR将在肿瘤微环境中被稳健地激活，并且即使在肿瘤细胞异质性存在的情况下也将导致强的抗肿瘤应答。这是未来肿瘤基质免疫靶向的高风险/高回报概念，这一目标的目标将是建立可行性证据，并证明这种CAR可以由fibulin-3产生和特异性激活。该项目的成功完成将导致一系列针对GBM微环境的主要生物制剂，并具有不同水平的临床前验证。这些结果将用于推进最有希望的候选药物作为GBM的研究性新药提交。

 DESCRIPTION (provided by applicant): The goal of this project is to develop and validate novel biological reagents against glioblastomas (GBMs), which are one of the most common and the most aggressive kind of brain tumors. GBMs are highly invasive in brain tissue and their dispersion is exacerbated by cytotoxic and antiangiogenic therapies, resulting in a much worse clinical picture when the tumor recurs. Novel therapeutics addressing the biology of GBM should be able to disrupt invasion or vascularization and sensitize tumor cells, thus causing tumor disruption in multiple fronts when combined with conventional therapies. This project will focus on the protein fibulin-3 identified by our group, which is secreted by GBM cells and has unique localization and mechanisms in GBM but is absent from normal brain. Fibulin-3 is a novel activator of the Notch pathway and genetic targeting of this protein disrupts Notch signaling in the tumor and reduces GBM growth and resistance. We have produced a function-blocking antibody against fibulin-3 ("mAb428.2") and in this project we will use it as a starting point to generate and validate a family of anti-fibulin-3 recombinant reagents. In Specific Aim 1, we propose to validate safety and efficacy of mAb428.2 against intracranial GBMs to advance it towards investigational new drug status. We hypothesize that anti-fibulin-3 will reduce tumor growth and vascularization with no significant peripheral toxicity or local off-target effects. We will study mAb428.2 specificity, cross-reactivity, biodistribution, safety and anti-tumor efficacy following FDA guidelines, aiming at completing pre-IND validation. In Specific Aim 2, we propose to develop and validate an anti-fibulin-3 single-chain immunotoxin as cytotoxic agent for GBM. We have already generated a single-chain variable fragment (scFv) derived from mAb428.2 and hypothesize that the smaller size of this fragment will improve access and toxicity against GBM. We will conjugate the scFv with Pseudomonas exotoxin and will validate this novel immunotoxin for specificity, safety and efficacy against intracranial GBM. Finally, using fibulin-3 scFv, in Specific Aim 3 we propose to generate and test a chimeric antigen receptor (CAR) against fibulin-3. We hypothesize that a fibulin-3-binding CAR expressed in T lymphocytes will be robustly activated in the tumor microenvironment and will result in strong anti-tumor response even in presence of tumor cell heterogeneity. This is a high-risk/high-reward concept for future immunotargeting of the tumor stroma, and the goal of this aim will be to establish a proof of feasibility and demonstrate that such CAR can be generated and specifically activated by fibulin-3. Successful completion of this project will result in a family f lead biological agents targeting the GBM microenvironment, with different levels of preclinical validation. These results will be used to advance the most promising candidate(s) for submission as investigational new drug(s) for GBM.