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， 在几乎所有NSCLC肿瘤暴露的患者中， 酪氨酸激酶抑制剂（TKI）。奥希替尼，一种靶向EGFR-T790 M的不可逆EGFR抑制剂 突变型非小细胞肺癌的一线治疗，但耐药性在12-24个月后出现。的 检查点蛋白（PD 1，PDL 1）和层粘连蛋白B2（LMNB 2）与多种肿瘤的不良预后相关。 癌症。我们实验室的结果表明，在H1975肿瘤（表达L 858 R/T790 M-EGFR）中， 突变），在其他蛋白中，PDL 1和LMNB 2的下调可以显著减少肿瘤细胞的生长。 异种移植小鼠中的负荷（蛋白质组学分析。此外，来源于自然杀伤细胞的外来体（EV） 细胞（NK 92 MI，NKEV）含有各种细胞溶解蛋白，并显示出作为抗癌剂的潜力。 在我们的实验室中，我们观察到NKEV（使用含有IL-15的PBS生物反应器）显示40%的细胞杀伤 当与来自HEK或MSC细胞的EV相比时，在1X 101 t3颗粒的浓度下， 肺PDX细胞中15- 20%的细胞杀伤。此外，H1975抗性（H1975 R）异种移植肿瘤，当 用NKEV处理的小鼠下调HO 1、波形蛋白。NF-kB、P38 MAPK与对照组比较差异有显著性（P<0.001 以控制和HEK衍生的EV，这表明它们通过诱导细胞凋亡和其他可能的抗癌作用， 机制等此外，发现NKEVS以显著量有效地递送荧光mir 3133-TYE (as与对照相比）对H1975 R肿瘤的靶向作用，显示了它们的靶向潜力。 进一步，我们探索了调节LMNB 2（mir-3133）和PDL 1（mir 5193）的微小RNA，并表明 在体外和体外实验中，它们分别能显著下调LMNB 2和PDL 1的表达。 PDX肿瘤（TM 00199，杰克逊实验室），仅当在NSG小鼠中作为EV制剂递送时。因此，基于 根据我们强有力的初步数据，我们假设携带LMNB 2和PDL 1微小RNA的NKEV将 将其有效载荷递送至奥希替尼耐药NSCLC，并将能够通过在 与卡铂联合使用，副作用最小。为了验证这一假设，我们提出以下建议： 独立目标： 目的1：含有PDL 1和LMNB 2微小RNA的NK-EV的制剂，并在体外对它们进行评价。 与卡铂组合抗H1975（R和野生型）和PDX细胞 目的2：在H1975耐药中对双重micro-RNA NKEV进行毒理学和药效学评价 PDX模型该提案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的微RNA制剂，并了解它们在克服耐药性方面的作用， 将来申请R 01方案或1期临床研究。

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.