Adrenergic modulation of cellular immune functions in CAR T cell-induced cytokine release syndrome

使用表达肿瘤定向嵌合抗原受体（CAR）的T细胞（CART）的免疫疗法是一种快速的免疫治疗方法。 在抗CD 19 CART中观察到的一种新兴的、有前途的癌症治疗方法，可以引起快速、持久的临床反应。 （CART 19）治疗的⑶ 19 + B细胞恶性肿瘤。然而，CART引起的严重或致命的免疫相关不良反应， 包括麻黄碱释放综合征（CRS）在内的irAE是重要的临床障碍和安全性问题 这阻碍了CART在癌症患者中的使用，即使有严格的患者监测和支持性护理。当前工具 为了减轻这种免疫毒性/CRS，靶向单个细胞因子/受体（例如IL-6 R、IL-1 Ra、GM-CSF）或 非特异性免疫抑制，但预防和治疗这种毒性将需要更好地了解 CART诱导的CRS的细胞/分子介质。该项目旨在克服这些障碍， 更有效的免疫毒性干预，从而更有效的CART治疗， PI的新的，潜在的可翻译的发现，在塑造CART治疗中的关键作用的儿茶酚胺（CAT）- 相关免疫激活。我们发现，阻断CAT的合成， 在小鼠中，也增强了其肿瘤根除，还发现CAT水平在患有 CART诱发的CRS与症状和IL-6水平相关，表明CATs有助于人类CRS 病理生理学该项目的目标是确定这些新的CAT的机制CRS调节作用 作用及其对巨噬细胞和抗肿瘤反应的影响，并评估 使用人类患者样品改善CART癌症治疗结果。具体目标是：1）确定 介导α1-AR亚型及其在调节CART诱发的细胞因子释放和抗肿瘤反应中的作用 体外和体内，基于我们的发现，CAT相关的细胞因子释放是由α1-肾上腺素能介导的， 受体（α1-AR），亚型未知。我们将鉴定主要介导 使用体外共培养测定和小鼠CART，巨噬细胞和CART中CAT诱导的细胞因子产生 通过α1-AR亚型的基因敲除（KO）和敲低（KD）的治疗模型。2)确定影响 骨髓源性CAT对体外和小鼠CART治疗期间细胞因子释放和骨髓功能的影响 模型，通过酪氨酸羟化酶（TH）KO灭活骨髓细胞中的CAT合成，并评估其影响 在共培养试验中和体内使用LysM-Cre介导的TH对细胞因子释放和巨噬细胞功能的影响 KO小鼠。3)评估CART治疗期间人类患者的CAT诱导，以及CAT对irAE的双重影响 和肿瘤反应，以及CAT措施和阻滞剂在预测和预防CRS中的潜在效用， 定量在CART治疗期间收集的患者血清样品中的循环CAT和细胞因子水平，和 评估CAT水平与临床和实验室CRS指数、抗肿瘤反应和存活率的相关性。

Immunotherapy using tumor-directed chimeric antigen receptor (CAR)-expressing T cells (CART) is a rapidly- emerging, promising cancer therapy that can elicit rapid, durable clinical responses, seen in anti-CD19 CART (CART19)-treated CD19+ B-cell malignancies. However, CART-evoked severe or fatal immune-related adverse events (irAE) including cytokine-release syndrome (CRS) are significant clinical barriers and safety concerns that impede CART use in cancer patients, even with strict patient monitoring and supportive care. Current tools for mitigating such immunotoxicities/CRS target individual cytokines/receptors (e.g. IL-6R, IL-1Ra, GM-CSF) or nonspecific immunosuppression, but preventing and treating such toxicity will require better understanding of cellular/molecular mediators of CART-induced CRS. This project aims to overcome these barriers and enable more effective interventions against immunotoxicities, and thus more effective CART treatments, building upon the PIs' novel, potentially translatable findings of a key role of catecholamines (CATs) in shaping CART therapy- associated immune activation. We found that blocking CAT synthesis both reduced CRS during CART therapy in mice and also enhanced its tumor eradication, and also found that CAT levels are elevated in patients with CART-evoked CRS, and correlated with symptoms and IL-6 levels, suggesting CATs contribute to human CRS pathophysiology. The project's goal is to identify the mechanistic CRS-modulating roles of these novel CAT actions and their impacts on macrophages and anti-tumor responses, and assess translational potential for improving CART cancer therapy outcomes using human patient samples. Specific Aims are: 1) Determine the mediating α1-AR subtype and its role in modulating CART-evoked cytokine release and anti-tumor responses in vitro and in vivo, based on our finding that CAT-associated cytokine release is mediated by α1-adrenergic receptors (α1-AR), of as-yet unknown subtype(s). We will identify the α1-AR subtype(s) that mainly mediate(s) CAT-evoked cytokine production in macrophages and CART, using in vitro co-culture assays and a mouse CART therapy model via genetic knockout (KO) and knockdown (KD) of α1-AR subtypes. 2) Determine the impact of myeloid-derived CATs on cytokine release and myeloid function during CART-therapy in vitro and in a mouse model, by inactivating CAT synthesis in myeloid cells via tyrosine hydroxylase (TH) KO, and assess its impact on cytokine release and macrophage function in a co-culture assay and in vivo using a LysM-Cre-mediated TH KO mouse. 3) Assess CAT induction in human patients during CART therapy, and CATs' dual impacts on irAE and tumor responses, and potential utility of CAT measures and blockers in predicting and preventing CRS, by quantifying circulating CAT and cytokine levels in patient serum samples collected during CART therapy, and evaluating correlations of CAT levels with clinical and laboratory CRS indices, anti-tumor responses and survival.