Immunotherapy Modeling in Organoids Co-preserving Tumor and Infiltrating Immune Compartments

项目总结 免疫系统显著区分自我和非自我/自我异常抗原，提供 精致的抗肿瘤专一性和抑制肿瘤形成。然而，肿瘤免疫监控是 不幸的是，反对肿瘤细胞逃避免疫反应。免疫检查点封锁(ICB) 靶向PD-1、PD-L1、CD40等，以及过继细胞转移(CAR-T、批量TIL)有利于调节 为了治疗上的利益，这种平衡。然而，应答率往往是不完整的，进行性疾病是 常见的和可预测的生物标记物是次优的。 由于缺乏体外模型，下一代免疫疗法的发展一直受到阻碍。 这在功能上概括了肿瘤和浸润性免疫细胞之间的同基因相互作用。作为回应， 我们已经开发了一种有机方法，将原代人类肿瘤活检组织与其浸润性组织培养在一起。 免疫成分作为一个凝聚的单位，不需要重组。这些“病人衍生的肿瘤有机化合物”(PDO) 与内源性T、B、NK细胞和巨噬细胞一起保存肿瘤细胞，有力地重组T细胞 原始肿瘤的受体克隆型谱系，最重要的是，表现为肿瘤浸润性淋巴细胞(TIL) 抗PD-1/PD-L1治疗性抗体的扩增、激活和肿瘤细胞杀伤(Cell，2018)。 因此，PDO系统代表了人类肿瘤-免疫相互作用的整体器官模型。 在这里，我们利用PDO技术来研究免疫治疗机制和 PD-1反应性皮肤鳞状细胞癌和黑色素瘤的治疗 治疗前后的人体活检和小鼠模型。目标1假设检查点抑制 诱导复杂而有序的网络反应，涉及免疫-肿瘤和免疫-免疫串扰。 因此，AIM 1使用执行PDO的连续时间进程采样的能力来定义单细胞RNA-SEQ 抗PD-1早期抗肿瘤免疫应答的网络细胞串扰模型 急性时间点通常无法在几个月后进行临床活组织检查。重要的是，比较 在有反应的小鼠和无反应的小鼠和人类器官中的这种免疫繁殖将定义结节 给予抵抗的点数。目的2以PDO为活体改进块状TIL过继转移免疫治疗 通过抗PD-1检查点抑制来丰富肿瘤反应性小鼠和人黑色素瘤TIL的生物反应器， 随后对增强的体外和体内抗肿瘤活性进行了测试。最后，AIM 3进行协同治疗 比较治疗前活检CSCC PDO的抗PD-1反应与临床结果的试验。此外，邮寄- 用抗PD-1抗体和一种新型灭活PD-1的试剂再次挑战治疗活组织PDO 去磷酸化。因此，我们利用整体PDO模型来保护内源性肿瘤上皮和免疫 组件通过我们的Calvin Kuo(有机化合物)团队研究和改进癌症免疫治疗， Mark Davis和Chris Garcia(肿瘤免疫学)，Anne Chang和Dimitri Colevas(CSCC临床医生)。

PROJECT SUMMARY The immune system remarkably distinguishes between self and non-self/self-aberrant antigens, affording exquisite anti-tumor specificity and inhibition of tumorigenesis. However, tumor immunosurveillance is unfortunately opposed by tumor cell evasion of the immune response. Immune checkpoint blockade (ICB) targeting PD-1, PD-L1, CD40 and others, as well as adoptive cell transfer (CAR-T, bulk TILs) favorably modulate this equilibrium for therapeutic benefit. However, response rates are often incomplete, progressive disease is common, and predictive biomarkers are suboptimal. The development of next-generation immunotherapies has been hindered by a lack of in vitro models that functionally recapitulate syngeneic interactions between tumor and infiltrating immune cells. In response, we have developed organoid methods that culture primary human tumor biopsies together with their infiltrating immune components as a cohesive unit without reconstitution. These “patient-derived tumor organoids” (PDO) preserve tumor cells alongside endogenous T, B, NK cells and macrophages, robustly recapitulate the T cell receptor clonotype repertoire of the original tumor, and crucially, manifest tumor-infiltrating lymphocyte (TIL) expansion, activation and tumor cell killing in response to anti-PD-1/PD-L1 therapeutic antibodies (Cell, 2018). The PDO system thus represents a holistic organoid model of human tumor-immune interactions. Here, we leverage the PDO technique to investigate immunotherapeutic mechanisms and treatments in PD-1-responsive cutaneous squamous cell carcinoma (cSCC) and melanoma, exploiting pre- and post-treatment human biopsies and mouse models. Aim 1 hypothesizes that checkpoint inhibition induces a complex and sequential network response involving immune-tumor and immune-immune crosstalk. Thus, Aim 1 employs the ability to perform serial time-course sampling of PDOs to define a single cell RNA-seq network cellular crosstalk model of the early anti-PD-1-stimulated anti-tumor immune response over multiple acute time points typically inaccessible to clinical biopsies performed after months. Importantly, comparison of this immune propagation in responding versus non-responding mouse and human organoids will define nodal points conferring resistance. Aim 2 improves bulk TIL adoptive transfer immunotherapy by using PDOs as living bioreactors to enrich tumor-reactive mouse and human melanoma TILs by anti-PD-1 checkpoint inhibition, followed by testing of enhanced anti-tumor activity in vitro and in vivo. Lastly, Aim 3 performs a co-treatment trial comparing anti-PD-1 responses of pre-treatment biopsy cSCC PDOs to clinical outcomes. Further, post- treatment biopsy PDOs are re-challenged with anti-PD-1 and a novel agent inactivating PD-1 by dephosphorylation. We thus utilize the holistic PDO model preserving endogenous tumor epithelial and immune components en bloc to investigate and improve cancer immunotherapy via our team of Calvin Kuo (organoids), Mark Davis and Chris Garcia (tumor immunology) and Anne Chang and Dimitri Colevas (cSCC clinicians).