A PLATFORM TECHNOLOGY TO GENETICALLY REPROGRAM CANCER CELLS FOR ENHANCED IMMUNOTHERAPY

项目总结 癌症免疫治疗的进展在治疗难治性肿瘤方面具有巨大潜力 常规治疗，以及由天然或人工抗原提呈细胞(APC)体外激活的T细胞 靶向和杀伤癌细胞已在临床上被证明可以提高高度侵袭性肺癌患者的生存率 癌症。APC通常通过呈递肿瘤抗原特异性信号1来激活T细胞，该信号由主要的 组织相容性复合体(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；以及分泌信号3，用于招募和激活 免疫细胞。而不是设计患者的APC来引导T细胞对肿瘤或 制造人工合成的APC，这两个都是昂贵、复杂和/或患者特定的过程，我们 提议对癌细胞本身进行重新编程，使其成为肿瘤来源的APC(TAPC)。因为肿瘤 细胞已经固有地表达信号1(MHC I背景下的肿瘤抗原)，它们可以在 SITE表达其他必要的信号，因此充当APC，指导细胞毒性T细胞反应 对抗他们自己。MHC I低表达的肿瘤细胞将刺激自然杀伤(NK)细胞来辅助这一过程 目的。我们已经设计了合成的、非病毒的纳米颗粒，可以将DNA输送到高密度的癌细胞 在健康组织上的有效性和特异性，我们将把这些注射到肿瘤块中以诱导表达 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 癌症)和四个体外肿瘤模型作为例子。我们提议的实验将首先优化 用于体内和体外高表达和肿瘤特异性的纳米颗粒制剂。到时候我们会的 显示癌细胞重新编程表达信号2和3后的活化和T和NK细胞 以及体外和体内的信号1。最后，我们将在肿瘤内注射后显示抗癌效果。 这些纳米颗粒在免疫活性小鼠癌症模型中的作用，重点是转移模型，以及 检查我们技术背后的免疫学机制。重要的是，因为我们的目标是使用 DNA传递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 通过基因转移黑色素瘤细胞，我们的策略只需要具有代表性的恶性细胞亚群 成功地进行了转基因。如果成功，这可能导致负担得起的、完全合成的、局部的、抗原不可知的 尽管如此，治疗仍会导致肿瘤的抗原特异性全身免疫排斥反应。

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.