IL-15 and -21 armored GPC3-specific CAR T cells for children with solid tumors

CAR T细胞可以在血液癌患者中诱导显著的抗肿瘤反应，但已经被证实是一种免疫抑制剂。 对实体瘤患者基本无效。几个障碍限制了CAR T在实体恶性肿瘤中的疗效 包括有限数量的安全实体瘤特异性抗原和免疫抑制性肿瘤 微环境（TME）缺乏支持刺激。此外，目前还不清楚CAR T细胞如何 影响人类TME中免疫逃逸机制的进化。我们开发并测试了一套 嵌合抗原受体（汽车），其1）靶向磷脂酰肌醇蛋白聚糖-3（GPC 3），一种由选择性表达的肿瘤抗原， 几种儿科实体瘤但非非恶性组织，和2）共表达白细胞介素-15（IL 15）和IL 21， 对T细胞存活、扩增、持久性和抗肿瘤效应物保留重要的细胞因子 功能.我们发现，共表达第二代GPC 3-CAR和IL 15（15.GPC3-CAR）的T细胞在体外表达。 与GPC 3-CAR T细胞相比，具有显著改善的扩增、持久性和抗肿瘤活性，并且 IL 15和IL 21（15.21.GPC3-CAR）的共表达进一步增强了这些临床前特性， 模型此外，我们的临床前研究表明，15.21.GPC3-CAR T细胞独特地维持表达， T细胞因子-1，它保护CAR T细胞免于耗竭，并保留增殖能力， 重复的GPC 3介导的激活。由于这些表达精氨酸的CAR的安全性和有效性参数 由于人类中的T细胞是未知的，因此我们建议在人类中评估15.GPC3-CAR和15.21.GPC3-CAR T细胞。 在复发性或难治性儿童中使用贝叶斯最佳间隔剂量递增的序贯I期研究 GPC 3阳性实体瘤。我们的多学科团队已经产生了有希望的安全性和有效性 结果用单独表达GPC 3-CAR的T细胞治疗儿童。我们假设15.GPC3-CAR和 15.21.GPC3-CAR T细胞将是安全的，并且IL 15与IL 21的共表达将使GPC 3-CAR T细胞能够与IL 21共表达。 克服抑制性TME，导致稳健的扩增、持久性和持久的抗肿瘤反应。在 目的1，我们将评估15.GPC3-CAR和15.21.GPC3-CAR T细胞在GPC 3阳性儿童中的安全性。 实体瘤在目标2中，我们将确定CAR T细胞的体内持久性和抗肿瘤活性。最后在 目的3，通过评估CAR T细胞产物、外周血样本和输注后肿瘤 活检，我们将定义GPC 3-CAR T细胞，癌细胞和非GPC 3-CAR T细胞的转录组学和表型谱。 肿瘤基质细胞我们将确定CAR T细胞中的生存程序并定义免疫逃逸 使用单细胞RNA测序、流式细胞术和COdetection-by-inDEXing来研究TME中的机制。 通过拟议的研究，我们将确定安全性、体内扩展和持久性以及抗肿瘤性。 我们的治疗细胞的活性。结果将确定CAR T细胞和免疫逃逸中的生存程序 人类TME中的机制，为制定有效措施提供蓝图， 基于CAR和其他免疫疗法用于实体瘤患者。

CAR T cells can induce remarkable antitumor responses in patients with hematologic cancers but have been largely ineffective in patients with solid tumors. Several barriers limit CAR T efficacy in solid malignancies including the limited number of safe solid tumor-specific antigens and an immunosuppressive tumor microenvironment (TME) that lacks supporting stimuli. Furthermore, it remains unknown how CAR T cells influence the evolution of immune escape mechanisms in the human TME. We developed and tested a set of chimeric antigen receptors (CARs) that 1) target glypican-3 (GPC3), a tumor antigen selectively expressed by several pediatric solid tumors but not non-malignant tissues, and 2) co-express interleukin-15 (IL15) and IL21, cytokines that are important for T cell survival, expansion, persistence, and retention of antitumor effector functionality. We found that T cells co-expressing a second generation GPC3-CAR and IL15 (15.GPC3-CAR) have significantly improved expansion, persistence, and antitumor activity compared to GPC3-CAR T cells, and that co-expression of both IL15 and IL21 (15.21.GPC3-CAR) further enhances these properties in preclinical models. Additionally, our preclinical studies showed that 15.21.GPC3-CAR T cells uniquely maintain expression of T cell factor-1, which protects CAR T cells from exhaustion and preserves proliferative capacity following repeated GPC3-mediated activation. Since the safety and efficacy parameters of these cytokine-expressing CAR T cells in humans are unknown, we propose to evaluate 15.GPC3-CAR and 15.21.GPC3-CAR T cells in sequential phase 1 studies using Bayesian Optimal Interval dose escalation in children with relapsed or refractory GPC3-positive solid tumors. Our multidisciplinary team has already generated promising safety and efficacy results treating children with T cells expressing the GPC3-CAR alone. We hypothesize that 15.GPC3-CAR and 15.21.GPC3-CAR T cells will be safe and that co-expression of IL15 with IL21 will enable GPC3-CAR T cells to overcome the inhibitory TME, resulting in robust expansion, persistence, and durable antitumor responses. In Aim 1, we will evaluate the safety of 15.GPC3-CAR and 15.21.GPC3-CAR T cells in children with GPC3-positive solid tumors. In Aim 2, we will determine the in vivo persistence and antitumor activity of CAR T cells. Finally, in Aim 3, through the evaluation of CAR T cell products, peripheral blood samples, and post-infusion tumor biopsies, we will define the transcriptomic and phenotypic profiles of GPC3-CAR T cells, cancer cells, and non- neoplastic stromal cells. We will identify survival programs in CAR T cells and define immune escape mechanisms in the TME using single-cell RNA sequencing, flow cytometry, and COdetection-by-inDEXing. Through the proposed research, we will determine the safety, in vivo expansion and persistence, and antitumor activity of our therapeutic cells. The results will identify survival programs in CAR T cells and immune escape mechanisms in the human TME to provide a blueprint for developing effective measures that boost the efficacy of CAR-based and other immunotherapies for patients with solid tumors.