Arming Oncolytic HSV Vectors to Induce Anti-GBM Immune Responses in Syngeneic Mice

溶瘤HSV载体（oHSV）在多形性胶质母细胞瘤（GBM）的治疗中提供了相当大的希望。 在以前的研究中，我们设计了一个新的基础载体（KGN-4：T124），该载体在正常脑中被阻断复制 通过脑特异性细胞miR-124靶向ICP 4表达，这个基础载体被进一步重新定位 （KGNE-4：T124）以实现表达EGFR/EGFRvIII的GBM细胞的选择性感染。准备好这个向量 基质金属蛋白酶（KGNE-4：T124-MMP 9）增强载体在异种细胞中的扩散和溶瘤作用 动物模型然而，单独的病毒裂解不太可能实现肿瘤细胞的完全消除， 复发有效消除所有肿瘤细胞将需要诱导先天性和适应性抗肿瘤 免疫力不幸的是，GBM下调了维持免疫激活所需的细胞机制。 细胞，包括NK、巨噬细胞和细胞毒性T细胞。因此，我们建议测试假设，GBM 用我们的oHSV基础载体KGN-4：T124和KGNE-4：T124的治疗可以通过载体武装来增强 具有抑制肿瘤进展并诱导先天性和适应性抗肿瘤免疫的免疫调节基因 应答为此，我们将首先利用来源于GBM肿瘤的肿瘤细胞系（BAGL 1），该肿瘤细胞系是在GBM中诱导的。 BALB/c小鼠通过睡美人转座编码荧光素酶、抗p53 shRNA、N-RasV 12和 人EGFRvIII。这些实验将扩展到由Dr. 埃里克·霍兰德避免了肿瘤细胞注射。我们的目标将寻求对抗免疫逃避的三个方面 在GBM中：（i）ADAM 17介导的NK细胞活化剂脱落和NK功能降低，（ii） 导致NK和T细胞耗竭的免疫检查点分子，以及（iii）M2巨噬细胞的存在 这有助于免疫抑制肿瘤微环境（TME）。在目标1中，我们将检验假设 我们的非武装基础载体，统称为KGN（E）-4：T124，将提供有效的GBM 在免疫活性小鼠中的治疗。我们将确定载体给药对肿瘤内载体的影响。 分布、TME组成和动物存活率，并评估EGFR靶向的价值。我们亦会研究 既往HSV免疫对治疗结果的影响。在目标2中，我们将检验KGN（E）- 4：装备有TIMP-3（ADAM 17抑制剂/VEGF-R2拮抗剂）的T124衍生物将改善治疗。我们将 评估TIMP-3表达对NK细胞活化、肿瘤扩散和动物存活的影响。在目标3中， 将检验装备有单链抗体（scFv）检查点抑制剂（抗PD-L1或抗PD-L2）的载体的假设。 抗-CTLA 4），单独或与免疫抑制性（M2）TAM群体的消耗组合， BLZ 945介导的抑制集落刺激因子-1受体（CSF-1 R），将产生有效的抗肿瘤作用 免疫，特别是在HSV免疫小鼠中。将使用双侧肿瘤建立抗肿瘤免疫 模型，并通过选择性抗体介导的T细胞耗竭证实。

Oncolytic HSV vectors (oHSV) offer considerable promise in the treatment of Glioblastoma Multiforme (GBM). In previous studies we designed a new base vector (KGN-4:T124) that is blocked for replication in normal brain tissue by brain-specific cellular miR-124 targeting of ICP4 expression. This base vector was further retargeted (KGNE-4:T124) to achieve selective infection of GBM cells expressing EGFR/EGFRvIII. Arming of this vector with a matrix metalloproteinase (KGNE-4:T124-MMP9) enhanced vector spread and oncolysis in xenogeneic animal models. However, virolysis alone is unlikely to achieve complete elimination of tumor cells that can cause recurrence. Effective elimination of all tumor cells will require the induction of innate and adaptive anti-tumor immunity. Unfortunately, GBM down-regulates the cellular machinery needed to sustain activation of immune cells including NK, macrophages and cytotoxic T cells. Therefore we propose to test the hypothesis that GBM treatment with our oHSV base vectors, KGN-4:T124 and KGNE-4:T124, can be enhanced through vector arming with immunomodulatory genes that inhibit tumor progression and induce innate and adaptive anti-tumor immune responses. To this end, we will initially exploit a tumor cell line (BAGL1) derived from a GBM tumor induced in BALB/c mice by Sleeping Beauty transposition of genes encoding luciferase, anti-p53 shRNA, N-RasV12 and human EGFRvIII. These experiments will be extended to a genetically-induced GBM model developed by Dr. Eric Holland that avoids tumor cell injection. Our aims will seek to counteract three aspects of immune evasion in GBM: (i) ADAM17-mediated shedding of NK cell activators and reduced NK function, (ii) expression of the immune checkpoint molecules that lead to NK and T cell exhaustion, and (iii) the presence of M2 macrophages that contribute to an immunosuppressive tumor microenvironment (TME). In Aim 1, we will test the hypothesis that our unarmed base vectors, collectively referred to as KGN(E)-4:T124, will provide an effective GBM treatment in immune competent mice. We will determine the impact of vector dosing on intratumoral vector distribution, TME composition and animal survival, and assess the value of EGFR targeting. We will also examine the effect of prior HSV immunization on treatment outcomes. In Aim 2, we will test the hypothesis that KGN(E)- 4:T124 derivatives armed with TIMP-3 (ADAM17 inhibitor/VEGF-R2 antagonist) will improve therapy. We will evaluate the impact of TIMP-3 expression on NK cell activation, tumor spread and animal survival. In Aim 3, we will test the hypothesis that vectors armed with single chain antibody (scFv) checkpoint inhibitors (anti-PD-L1 or anti-CTLA4), alone or in combination with depletion of the immunosuppressive (M2) TAM population using BLZ945-mediated inhibition of colony-stimulating factor-1 receptor (CSF-1R), will produce effective anti-tumor immunity, particularly in HSV immune mice. Anti-tumor immunity will be established using a bi-lateral tumor model and confirmed by selective antibody-mediated T cell depletion.