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

项目概要 免疫系统可以显着区分自身抗原和非自身/自身异常抗原，从而提供 精湛的抗肿瘤特异性和抑制肿瘤发生。然而，肿瘤免疫监视 不幸的是，肿瘤细胞逃避了免疫反应。免疫检查点阻断 (ICB) 针对 PD-1、PD-L1、CD40 等以及过继细胞转移（CAR-T、批量 TIL）进行有利调节 这种治疗益处的平衡。然而，缓解率往往不完全，疾病进展是 常见的生物标志物和预测性生物标志物均不理想。 缺乏体外模型阻碍了下一代免疫疗法的发展 在功能上概括了肿瘤和浸润免疫细胞之间的同基因相互作用。作为回应， 我们开发了类器官方法，可培养原发性人类肿瘤活检及其浸润 免疫成分作为一个有凝聚力的单位而无需重组。这些“患者来源的肿瘤类器官”（PDO） 将肿瘤细胞与内源性 T、B、NK 细胞和巨噬细胞一起保存，有力地再现 T 细胞 原发肿瘤的受体克隆型库，最重要的是，表现出肿瘤浸润淋巴细胞（TIL） 对抗 PD-1/PD-L1 治疗性抗体的反应导致肿瘤细胞的扩增、激活和杀伤（Cell，2018）。 因此，PDO 系统代表了人类肿瘤-免疫相互作用的整体类器官模型。 在这里，我们利用 PDO 技术来研究免疫治疗机制并 PD-1 反应性皮肤鳞状细胞癌 (cSCC) 和黑色素瘤的治疗，利用 治疗前和治疗后的人体活检和小鼠模型。目标 1 假设检查点抑制 诱导涉及免疫-肿瘤和免疫-免疫串扰的复杂且连续的网络反应。 因此，目标 1 利用对 PDO 进行连续时间过程采样的能力来定义单细胞 RNA-seq 早期抗PD-1刺激的抗肿瘤免疫反应的网络细胞串扰模型 几个月后进行的临床活检通常无法获得急性时间点。重要的是，比较 这种在有反应与无反应的小鼠和人类类器官中的免疫传播将定义节点 赋予阻力的点。目标 2 通过使用 PDO 作为活体来改善批量 TIL 过继转移免疫疗法 通过抗 PD-1 检查点抑制来丰富肿瘤反应性小鼠和人类黑色素瘤 TIL 的生物反应器， 随后测试增强的体外和体内抗肿瘤活性。最后，Aim 3 进行协同处理 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，后 治疗活检 PDO 再次受到抗 PD-1 和一种新型灭活 PD-1 药物的攻击 去磷酸化。因此，我们利用整体 PDO 模型保留内源性肿瘤上皮和免疫 通过我们的 Calvin Kuo（类器官）团队研究和改进癌症免疫疗法， Mark Davis 和 Chris Garcia（肿瘤免疫学）以及 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).