Engineering In Vivo Chimeric Antigen Receptor Macrophages (CARMs) using mRNA-exosomes for Cancer Immunotherapy

项目摘要 嵌合抗原受体（CAR）T细胞疗法已经彻底改变了血液病的治疗。 癌的然而，对于实体瘤，CAR T细胞面临挑战，包括肿瘤内异质性、动态变化、肿瘤细胞的增殖和分化。 靶受体的表达，以及T细胞通常无法运输到肿瘤以介导所需的免疫应答。 抗肿瘤作用与缺乏T细胞浸润相反，许多实体瘤具有丰富的免疫调节功能。 抑制性骨髓细胞，包括巨噬细胞。因此，将这些免疫抑制细胞转化为 杀肿瘤表型代表了基于细胞的治疗的一种有前途的策略。现在人们对 产生CAR巨噬细胞，其中自体巨噬细胞用由病毒递送的CAR转导， 离体载体以增强其吞噬作用、抗原呈递和细胞因子产生能力 再输注后。然而，CAR巨噬细胞的离体制备是复杂的、耗时的并且由于缺乏足够的细胞毒性，因此需要在体外制备CAR巨噬细胞。 与巨噬细胞的非分裂性质相比，通常是低效的。随着最近基于mRNA的 治疗，现在有可能在体内重新编程特定的免疫细胞群，从而消除免疫缺陷。 自体CAR细胞的复杂离体产生。我们目前的建议旨在提出一个创新的 使用装载mRNA的外泌体治疗HER2受体在体内产生CAR巨噬细胞的策略 乳腺癌阳性。这将是第一项评估在大肠杆菌中产生CAR巨噬细胞的可行性的研究。 体内使用mRNA递送平台并评估CAR巨噬细胞对癌症的抗肿瘤功效 免疫疗法我们假设我们的策略代表了一种革命性的生产CAR的方法， 巨噬细胞体内使用CAR mRNA加载的外泌体，并为细胞治疗提供了有前途的新方法 对抗实体瘤我们之前的研究表明，我们可以有效地生产载有mRNA的外泌体， 恢复实体瘤中的蛋白质表达。此外，我们的初步实验表明， 负载HER2 CAR mRNA的外泌体可以在体内产生CAR巨噬细胞， 功能协调发展的我们目前的研究将通过使用以下具体目标来测试我们的总体假设。目标1： 将使用CAR mRNA外泌体评估体内CAR巨噬细胞产生的动力学和毒性。在 目的2，我们将评估体内产生的CAR巨噬细胞的转录组学和功能谱，最后， 在目的3中，我们将评估体内产生的CAR巨噬细胞对鼠和鼠巨噬细胞的抗肿瘤作用。 表达人HER2的乳腺癌。如果成功，我们提出的研究可以克服一个主要的技术问题， 这是目前细胞治疗面临的最大障碍。mRNA外泌体平台可能会扩展到其他领域 CAR构建并极大地扩展了乳腺癌和其他实体癌细胞治疗的潜在效用。

PROJECT SUMMARY Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of hematologic cancers. However, for solid tumors, CAR T cells face challenges including intratumor heterogeneity, dynamic expression of target receptors, and often the inability for T cells to traffic to tumors to mediate the desired antitumor effect. In contrast to the lack of T cell infiltrates, many solid tumors are abundant in immune suppressive myeloid cells including macrophages. Therefore, converting these immune suppressive cells into tumoricidal phenotype represents a promising strategy for cell-based therapy. There are now strong interest in generating CAR macrophages in which autologous macrophages are transduced with CAR delivered by viral vectors ex vivo to enhance their phagocytosis, antigen presentation and cytokine producing capabilities following re-infusion. However, ex vivo preparation of CAR macrophages is complex, time consuming, and due to the non-dividing nature of macrophages, is often inefficient. With the recent advances in mRNA-based therapeutics, it is now possible to reprogram specific immune cell populations in vivo, thus eliminating the complex ex vivo production of autologous CAR cells. Our present proposal aims to propose an innovative strategy of generating CAR macrophages in vivo using mRNA-loaded exosomes to treat HER2 receptor positive breast cancer. This will be the first study to evaluate the feasibility of producing CAR macrophages in vivo using mRNA delivery platforms and assessing the antitumor efficacy of CAR macrophages for cancer immunotherapy. We hypothesize that our strategy represents a revolutionary way to produce CAR macrophages in vivo using CAR mRNA-loaded exosome and offers a promising new approach for cell therapy against solid tumors. Our previous study showed that we can efficiently produce mRNA-loaded exosomes to restore protein expression in solid tumors. Furthermore, our preliminary experiments showed that the exosomes loaded with HER2 CAR mRNA can produce CAR macrophages in vivo with enhanced effector functions. Our current study will test our overall hypothesis by using the following specific aims. In Aim 1, we will evaluate the dynamics and toxicity of CAR macrophage production in vivo using CAR mRNA exosomes. In Aim 2, we will evaluate transcriptomic and functional profiles of in vivo generated CAR macrophages, Finally, in Aim 3, we will assess the antitumor effect of in vivo generated CAR macrophages against both murine and human HER2 expressing breast cancer. If successful, our proposed research can overcome a major technical hurdle that is currently facing cell therapy. The mRNA exosome platform could potentially be expanded to other CAR constructs and greatly expand the potential utility of cell therapy for breast and other solid cancers.