Exosomal Based micro RNA delivery for Resistant Lung Cancer

摘要 非小细胞肺癌(NSCLC)是所有癌症中预后最差的，经过一段时间的 反应性、获得性耐药(例如，T790M突变)发生在几乎所有暴露的非小细胞肺癌肿瘤中 酪氨酸激酶抑制剂(TKI)。以EGFR-T790M为靶点的不可逆EGFR抑制剂奥西莫替尼 突变是突变非小细胞肺癌的一线治疗方法，但12-24个月后就会产生耐药性。这个 检查点蛋白(PD1、PDL1)和层蛋白B2(LMNB2)在多种情况下与预后不良有关 癌症。我们实验室的结果表明，在H1975肿瘤(表达L858R/T790M-EGFR)中 突变)，下调PDL1和LMNB2等蛋白质，可以显著减少肿瘤 异种移植小鼠的负担(蛋白质组学分析。此外，来自自然杀手的Exosome(EV) 细胞(NK92MI、NKEV)含有多种细胞溶解蛋白，已显示出潜在的抗癌作用。 在我们的实验室中，我们观察到NKEV(使用带有IL-15的PBS生物反应器)显示出40%的细胞杀伤率 当颗粒浓度为1×1010时，与来自HEK或MSC细胞的EVS相比，显示 对肺PDX细胞的杀伤率为15-20%。此外，当H1975耐药(H1975R)异种移植瘤时 经NKEV处理后，HO1、Vimentin表达下调。核因子-kB、p38MAPK表达显著高于对照组(P&lt；0.001)。 对照和HEK来源的EV提示其通过诱导细胞凋亡和其他可能的作用而发挥抗癌作用 机械装置。此外，还发现NKEV有效地传递了大量的荧光mir3133-TYe (与对照组相比)静脉给药时，H1975R肿瘤显示出其靶向潜力。 进一步研究了调控LMNB2(mir-3133)和PDL1(Mir5193)的microRNA，结果表明 它们在体外能显著下调LMNB2和PDL1的表达，在体外也能显著下调LMNB2和PDL1的表达。 PDX肿瘤(TM00199，Jackson实验室)，仅当作为EV制剂在NSG小鼠中提供时。因此，基于 根据我们强大的初步数据，我们假设携带LMNB2和PDL1微RNA的NKEV将 将他们的有效载荷传递给耐奥西莫替尼的非小细胞肺癌，并将能够通过使用 与卡铂联合使用，副作用最小。为了检验这一假设，我们提出了以下建议 独立目标： 目的1：构建含PDL1和LMNB2微RNA的NK-EVS，并对其进行体外评价 卡铂联合抗H1975(R和野生型)和PDX细胞 目的2：双微RNA NKEV对H1975耐药的毒理学和药效学评价 和PDX型号。这项提议的长期目标是用生物反应器产生足够的临床前数据 制造NKEV微型RNA配方，并了解它们在克服耐药性方面的作用，以便 在未来申请R01提案或第一阶段临床研究。

Abstract Non-small cell lung cancers (NSCLC) have the poorest outcome of all the cancers and after a period of responsiveness, acquired resistance (e.g. T790M mutation) occurs in virtually all NSCLC tumors exposed to Tyrosine Kinase Inhibitors (TKI). Osimertinib, an irreversible EGFR inhibitor which targets EGFR- T790M mutations is the first line treatment for mutated NSCLC but resistance develops after 12-24 months. The checkpoint proteins (PD1, PDL1) and laminB2 (LMNB2) are associated with a poor prognosis in a variety of cancers. Results from our laboratory have shown that in H1975 tumors (expressing L858R/T790M-EGFR mutations), downregulation of PDL1 and LMNB2, among other proteins, could significantly reduce tumor burden in xenotransplanted mice (proteomic analysis. Further, exosomes (EVs) derived from Natural killer cells (NK92MI, NKEVs) contain various cytolytic proteins and have shown potential as anticancer agents. In our laboratory, we observed that NKEVs (using a PBS bioreactor with IL-15), showed 40 percent cell kill at concentration of 1X1010 particles when compared to EVs derived from HEK or MSC cells which showed 15-20 percent cell kill in lung PDX cells. Further, H1975 resistant (H1975R) xenotransplanted tumors when treated with NKEVs downregulated HO1, vimentin. NF-kB, P38MAPK significantly (P<0.001) as compared to control and HEK derived EVs suggesting their anticancer role via inducing apoptosis and other possible mechanisms. Also, NKEVS were found to deliver fluorescent mir3133-TYE effectively in significant amounts (as compared to control) to H1975R tumors when given intravenously showing their targeting potential. Further we explored the micro-RNA which regulate LMNB2(mir-3133) and PDL1(mir5193) and showed that they could significantly downregulate the expression of LMNB2 and PDL1 respectively in vitro and also in PDX tumors (TM00199, Jackson labs), only when delivered as EV formulations in NSG mice. Hence based on our strong preliminary data, we hypothesize that NKEVs carrying LMNB2 and PDL1 micro RNA will deliver their payload to osimertinib resistant NSCLC and will be able to overcome resistance by using in combination with carboplatin with minimal side effects. To test this hypothesis, we propose the following independent Aims: Aim 1: Formulation of NK-EVs containing PDL1 and LMNB2 micro-RNA and evaluating them in vitro in combination with carboplatin against H1975 (R and wild type) and PDX cells Aim 2: Toxicological and Pharmacodynamic evaluation of the dual micro-RNA NKEVs in H1975 resistant and PDX models. The long-term goal of this proposal is to generate enough preclinical data with bioreactor manufactured NKEVs micro RNA formulations and to understand their role in overcoming resistance so as to apply for a R01 proposal or Phase 1 clinical studies in the future.