Naturally Targeted Exosomal TLR7/8 Agonist for Immunotherapy of Medulloblastoma

自然靶向外体TLR7/8激动剂免疫治疗髓母细胞瘤 尽管接受积极和剧毒的治疗，近一半被诊断为髓母细胞瘤的儿童将 死于复发性疾病。幸存者往往会留下与治疗相关的致残脑损伤。更多 需要有效和毒性较低的治疗方法来改善受... 髓母细胞瘤。Sonic Hedgehog(SHH)髓母细胞瘤富含肿瘤相关巨噬细胞 (TAM)，表达Toll样受体(TLR)7和8。重新编程这些TAM可能导致 免疫抑制签名和杀瘤和促炎签名的获得。我们已经展示了 TLR7/8激动剂Resiquimod包裹在聚合物纳米胶束中显著延长了G-1细胞的存活时间。 血脑屏障完整的SHH髓母细胞瘤基因工程小鼠模型(GEMM)的建立 (BBB)和肿瘤微环境(TME)。这种游离药物在G-Smo小鼠髓母细胞瘤模型中没有活性。 虽然胶束增加了这种药物对大脑毛细血管的溶解度和生物利用度，但它们并不针对 BBB。此外，胶束是动态的结构，在到达之前会在外围释放其大部分货物 大脑，这可能会降低治疗潜力并增加这种形式的副作用。因此，我们建议 以单核/巨噬细胞来源的外切体作为利奎莫特的天然靶向载体的不同处方 治疗髓母细胞瘤。我们发现，巨噬细胞分泌的外切体被运输到炎症患者体内。 并携带治疗分子穿过完好的血脑屏障。我们的初步数据显示巨噬细胞- 衍生的外切体在G-Smo小鼠的大脑中积累。受这些发现的启发，我们寻求开发 Exosome递送Requimod是治疗髓母细胞瘤的一种新方法。为了实现这一目标，我们开发了 外体掺入形式的requimod(exo-res)，它被证明使髓系细胞极化为亲 炎症性M1样表型。我们将测试以下假设：(A)在G-Smo小鼠体内释放外切体 髓母细胞瘤模型将有效地将Resiquimod输送到肿瘤，(B)输送的Resiquimod将 在髓母细胞瘤中重新极化TAMS，(C)这种新的靶向体外治疗方式将抑制肿瘤 与未靶向的第一代纳米胶束相比，生长和改善临床相关结果，POX- Res PM。我们的具体目标将是：1)确定外切体是否改善了Resiquimod的肿瘤分布 给带髓母细胞瘤的小鼠注射。SA2)确定外源性激素是否增强了抗肿瘤疗效 在髓母细胞瘤荷瘤小鼠中的利奎莫德。如果成功，这将导致新的治疗方法，有可能 改进髓母细胞瘤的治疗，用现有的放射和化疗方法取代 毒性更小，效果更好。

Naturally Targeted Exosomal TLR7/8 Agonist for Immunotherapy of Medulloblastoma Despite aggressive and highly toxic treatment, nearly half of the children diagnosed with medulloblastoma will die from recurrent disease. Survivors are often left with disabling treatment-associated brain injury. More effective and less toxic therapies are needed to improve clinical outcomes for children affected by medulloblastoma. Sonic Hedgehog (SHH) medulloblastoma is enriched in tumor associated macrophages (TAMs) which express Toll-like receptor (TLR) 7 and 8. Reprogramming these TAMs could result in the loss of immunosuppressive signatures and acquisition of tumoricidal and proinflammatory signatures. We have shown that TLR7/8 agonist, resiquimod, encapsulated in polymeric nano-micelles significantly prolonged survival in G- Smo mice - genetically engineered mouse model (GEMM) of SHH medulloblastoma with intact blood brain barrier (BBB) and tumor microenvironment (TME). The free drug was not active in G-Smo mice medulloblastoma model. While micelles increase solubility and bioavailability of this agent to the brain capillaries, they do not target the BBB. Moreover, micelles are dynamic structures and release most of their cargo peripherally before reaching the brain, which can decrease therapeutic potential and increase side effects of this format. Hence, we propose a different formulation using monocyte/macrophage derived exosomes as natural targeted carriers of resiquimod for medulloblastoma. We discovered that exosomes secreted from macrophages are transported to the inflamed brain and carry therapeutic molecules across the intact BBB. Our preliminary data suggest that macrophage- derived exosomes accumulate in the brain of the G-Smo mice. Inspired by these discoveries we seek to develop exosome delivered resiquimod as a novel approach to medulloblastoma. Toward this goal we developed the exosome incorporated form of resiquimod (exo-Res) that was shown to polarize myeloid cells toward pro- inflammatory M1-like phenotype. We will test the hypotheses that (a) exo-Res exosomes in a G-Smo mouse model of medulloblastoma will effectively deliver the resiquimod to the tumor, (b) the delivered resiquimod will repolarize TAMs in medulloblastoma, (c) this novel targeted exo-Res therapeutic modality will inhibit the tumor growth and improve the clinically relevant outcomes compared to untargeted first generation nano-micelles, POx- Res PMs. Our Specific Aims will be: 1) Determine if exosomes improve the tumor distribution of resiquimod administered to medulloblastoma-bearing mice. SA2) Determine if exo-Res enhances the anti-tumor efficacy of resiquimod in medulloblastoma-bearing mice. If successful, this will lead to novel therapy that has potential to improve medulloblastoma treatment by replacing the current radiation and chemotherapy with the one that is less toxic and more effective.