A PLATFORM TECHNOLOGY TO GENETICALLY REPROGRAM CANCER CELLS FOR ENHANCED IMMUNOTHERAPY

项目摘要 癌症免疫疗法的进展对于治疗难治性肿瘤具有巨大潜力。 常规治疗，以及通过天然或人工抗原呈递细胞（APC）离体引发的T细胞， 靶向和杀死癌细胞在临床上已经显示出提高高度侵袭性肿瘤患者的存活率， 癌的APC通常通过呈递肿瘤抗原特异性信号1来启动T细胞，该信号由主要的 组织相容性复合物（MHC）I分子与肿瘤抗原肽;共刺激信号2，指导 T细胞在识别肿瘤时的作用;和用于募集和激活T细胞的分泌信号3。 免疫细胞。不是设计患者的APC来指导T细胞对肿瘤的反应， 制造人工合成的APC，这两者都是昂贵的、复杂的和/或患者特异性的过程，我们 他们提出将癌细胞本身重新编程为肿瘤源性APC（tAPC）。因为肿瘤 细胞本身已经表达信号1（MHC I背景下的肿瘤抗原），它们可以被工程化， 原位表达其他必要的信号，因此作为APC，指导细胞毒性T细胞反应 反对他们自己。具有低MHCI表达的肿瘤细胞将刺激自然杀伤（NK）细胞来帮助这一点。 目的.我们设计了合成的非病毒纳米颗粒，可以将DNA以高浓度递送到癌细胞。 有效性和特异性，我们将这些注射到肿瘤块中，以诱导 信号2和信号3，使用两种不同的体内原位肿瘤模型（黑色素瘤和三阴性乳腺癌 癌症）和四种体外肿瘤模型作为实例。我们提出的实验将首先优化 用于体外和体内高表达和癌症特异性的纳米颗粒制剂。然后我们将 证明在癌细胞重编程以表达信号2和3后T和NK细胞的活化 沿着信号1在体外和体内。最后，我们将展示瘤内注射后的抗癌疗效 这些纳米颗粒在免疫活性鼠癌症模型中的作用，重点是转移模型， 研究我们技术背后的免疫机制重要的是，因为我们的目标是使用 DNA递送纳米颗粒刺激免疫系统杀死癌细胞，而不是直接杀死癌细胞。 通过基因转移转移黑色素瘤细胞，我们的策略只需要恶性细胞的代表性子集， 成功转染。如果成功，这可能会导致一个负担得起的，完全合成的，本地的，抗原不可知的， 然而导致肿瘤的抗原特异性全身免疫排斥的治疗。

PROJECT SUMMARY Advances in cancer immunotherapy have great potential for treating tumors that are refractory to conventional treatments, and T cells primed ex vivo by natural or artificial antigen-presenting cells (APCs) to target and kill cancer cells have been shown clinically to improve survival in patients with highly aggressive cancers. APCs normally prime T cells by presenting a tumor antigen-specific signal 1, consisting of a major histocompatibility complex (MHC) I molecule with a tumor antigen peptide; a co-stimulatory signal 2 that directs the action of the T cells upon recognition of the tumor; and a secreted signal 3 for recruitment and activation of immune cells. Instead of engineering the patient's APCs to direct a T-cell response against a tumor or fabricating artificial, synthetic APCs, both of which are costly, complex, and/or patient-specific processes, we propose to reprogram cancer cells themselves to become tumor-derived APCs (tAPCs). Because tumor cells already intrinsically express signal 1 (tumor antigen in the context of MHC I), they can be engineered in situ to express the other necessary signals and therefore act as APCs, directing cytotoxic T-cell responses against themselves. Tumor cells with low MHC I expression will stimulate natural killer (NK) cells to aid this purpose. We have designed synthetic, non-viral nanoparticles that can deliver DNA to cancer cells with high efficacy and specificity over healthy tissue, and we will inject these into a tumor mass to induce expression of signal 2 and signal 3, using two different in vivo orthotopic tumor models (melanoma and triple-negative breast cancer) and four in vitro tumor models as examples. Our proposed experiments will first optimize the nanoparticle formulation for high expression and cancer specificity in vitro and in vivo. We will then demonstrate activation and T and NK cells after reprogramming of cancer cells to express signals 2 and 3 along with signal 1 both in vitro and in vivo. Finally, we will show anti-cancer efficacy after intratumoral injection of these nanoparticles in immunocompetent murine cancer models, with a focus on models of metastasis, and examine the immunological mechanisms underlying our technology. Importantly, because our goal is to use DNA-delivery nanoparticles to stimulate the immune system to kill cancer cells, rather than directly killing the melanoma cells by gene transfer, our strategy requires only a representative subset of malignant cells to be successfully transfected. If successful, this could result in an affordable, fully synthetic, local, antigen-agnostic therapy that nevertheless leads to antigen-specific systemic immune rejection of tumors.