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

项目摘要 免疫系统显著区分自身和非自身/自身异常抗原， 具有良好的抗肿瘤特异性和抑制肿瘤发生的作用。然而，肿瘤免疫监视是 不幸的是，肿瘤细胞逃避免疫反应。免疫检查点阻断（ICB） 靶向PD-1、PD-L1、CD 40等，以及过继细胞转移（CAR-T，散装TIL）有利地调节 这种平衡是有益的。然而，反应率往往是不完全的，进展性疾病是 常见的和预测性的生物标志物是次优的。 由于缺乏体外模型，下一代免疫疗法的发展受到阻碍 其在功能上概括了肿瘤和浸润性免疫细胞之间的同源相互作用。作为回应， 我们已经开发出类器官方法，将原发性人类肿瘤活检组织与其浸润性细胞一起培养， 免疫成分作为一个没有重建的凝聚单位。这些“患者源性肿瘤类器官”（PDO） 与内源性T、B、NK细胞和巨噬细胞一起保存肿瘤细胞， 受体克隆型库的原始肿瘤，关键是，表现肿瘤浸润淋巴细胞（TIL） 抗PD-1/PD-L1治疗性抗体的扩增、活化和肿瘤细胞杀伤（Cell，2018）。 因此，PDO系统代表了人类肿瘤-免疫相互作用的整体类器官模型。 在这里，我们利用PDO技术来研究免疫机制， PD-1反应性皮肤鳞状细胞癌（cSCC）和黑色素瘤的治疗， 治疗前和治疗后的人类活检和小鼠模型。目的1假设检查点抑制 诱导涉及免疫-肿瘤和免疫-免疫串扰的复杂和顺序的网络响应。 因此，目标1采用进行PDO的连续时程采样的能力来定义单细胞RNA-seq 在多个时间点上的早期抗PD-1刺激的抗肿瘤免疫应答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 在应答与非应答小鼠和人类类器官中的这种免疫传播将定义淋巴结转移。 赋予抵抗力的点。目的2：以PDO为活细胞，提高TIL的过继免疫治疗效果 生物反应器通过抗PD-1检查点抑制富集肿瘤反应性小鼠和人黑素瘤TIL， 随后在体外和体内测试增强的抗肿瘤活性。最后，Aim 3执行共处理 比较治疗前活检cSCC PDO的抗PD-1应答与临床结局的试验。此外，后- 用抗PD-1和一种新的灭活PD-1的药物对治疗活组织检查的PDO进行再激发， 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 通过我们的卡尔文郭（类器官）团队， 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).