scFvPD1-FLT3 CAR T Cells for the Treatment of Relapsed Acute Myeloid Leukemia

项目摘要 急性髓系白血病(AML)在复发的老年患者中的中位生存期只有6个月。为了这个 因此，在降低发病率的同时提高疗效的靶向疗法受到了极大的关注。 嵌合抗原受体(CAR)T细胞靶向急慢性抗原表达的研究进展 淋巴细胞白血病揭示了基于免疫的治疗复发和/或难治性血液病的新方法 恶性肿瘤。然而，由于缺乏CAR T细胞，CAR T细胞尚未在临床上成功治疗AML 一个理想的靶点，因为一些已报道的靶点(如CD123和CD33)会耗尽造血干细胞和/或 髓系细胞。相反，我们的初步数据显示，用CAR T细胞靶向Flt3相对更安全 因为在体外，Flt3-CAR T细胞不会杀死健康的外周血单核细胞，而且不会显著 影响造血干细胞的自我更新和再繁殖。因此，我们认为Flt3是一个相对更好的目标 对于CAR T细胞在治疗AML中的作用。因此，我们产生了针对Flt3的CAR T细胞。Flt3-CAR T细胞 增强AML患者分离的AML细胞的体外清除和消除植入人AML细胞 免疫缺陷小鼠。T细胞(包括CAR T细胞)会耗尽并抑制免疫反应 当在肿瘤微环境中对抗肿瘤细胞时。这至少部分归因于表达的增加 PD-1受体，一种调节T细胞激活的关键检查点抑制物。抗PD-1 T细胞检查站 阻断抗体旨在逆转T细胞衰竭，并已显示出良好的临床疗效 治疗各种癌症。我们的团队和其他人之前已经证明，PD-1也表达 在癌症患者的NK细胞上，而在静止的NK细胞上不是。因此，将CAR T细胞和Car T细胞相结合更为有效。 PD-1检查点阻断以增强NK细胞和T细胞的能力。在这项研究中，我们添加了一个框架来 我们构建的表达抗PD-1单链抗体(ScFvPD1)的Flt3CAR。我们 已经用这种结构改造了T细胞，以产生所谓的scFvPD1-Flt3 CAR T细胞，能够 靶向Flt3的AML-CAR细胞同时表达可溶性ScFvPD1以恢复其功能 耗尽的T细胞。CAR T细胞和患者NK细胞也表达抑制性PD-1。因此，可溶性ScFvPD1 由scFvPD1-Flt3产生的CAR细胞将进一步增强Flt3-CAR T细胞在小鼠体内的抗肿瘤活性 内源性NK细胞和T细胞以旁分泌方式分泌。通过这种多管齐下的攻击来 增强对肿瘤细胞的免疫反应，我们相信我们的方法有潜力成功地治疗 复发和/或难治性AML。我们提出了三个目标来测试scFvPD1-Flt3 CAR细胞在体外和在 体内：目的1，进一步体外检测scFvPD1-Flt3 CAR T细胞；目的2，体内临床前检测scFvPD1- 目的3，使用scFvPD1-Flt3 CAR T细胞和米哚妥林联合治疗 最近被美国FDA批准，我们发现它可以诱导急性髓系白血病细胞表面表达Flt3。

Project Summary Acute Myeloid leukemia (AML) has a median survival of only six months in relapsed elderly patients. For this reason, there is great interest in developing targeted therapeutics to improve efficacy while decreasing morbidity. Recent reports of chimeric antigen receptor (CAR) T cells targeting antigens expressed on acute and chronic lymphocytic leukemia shed light on novel immune-based approaches for relapsed and/or refractory hematological malignancies. However, CAR T cells have not yet been successful in treating AML in the clinic due to the lack of an ideal target, as some reported targets (e.g., CD123 and CD33) deplete hematopoietic stem cells and/or myeloid cells. In contrast, our preliminary data showed that targeting FLT3 with CAR T cells is relatively safer because FLT3-CAR T cells do not kill healthy peripheral blood mononuclear cells in vitro and do not significantly affect self-renewal and repopulation of hematopoietic stem cells. Thus, we believe FLT3 is a relatively better target for CAR T cells in the treatment of AML. We therefore generated CAR T cells targeting FLT3. FLT3-CAR T cells enhance in vitro eradication of AML blasts isolated from patients and eliminate human AML cells engrafted into immunodeficient mice. T cells (including CAR T cells) can become exhausted and suppress immune responses when fighting tumor cells in the tumor microenvironment. This is at least partially due to the increased expression of the PD-1 receptor, a key checkpoint inhibitor modulating T cell activation. The anti-PD-1 T cell checkpoint blockade antibody is designed to reverse T cell exhaustion and has shown promising clinical efficacy for the treatment of various cancers. Our group and others have previously demonstrated that PD-1 is also expressed on NK cells in cancer patients but not in resting NK cells. Thus, it is more effective to combine CAR T cells and PD-1 checkpoint blockade to enhance the power of both NK cells and T cells. In this study, we added a frame to the FLT3 CAR construct that we have generated to express anti-PD-1 single chain antibodies (scFvPD1). We have engineered T cells with this construct to generate so-called scFvPD1-FLT3 CAR T cells that are able to target FLT3 in AML using FLT3-CAR T cells simultaneously expressing soluble scFvPD1 to restore the function of exhausted T cells. CAR T cells and patient NK cells also express inhibitory PD-1. Thus, the soluble scFvPD1 produced by scFvPD1-FLT3 CAR T cells will further augment the antitumor activity of FLT3-CAR T cells in an autocrine manner, and endogenous NK and T cells in a paracrine manner. With this multipronged attack to augment immune responses against tumor cells, we believe our approach has the potential to successfully treat relapsed and/or refractory AML. We have three Aims proposed to test scFvPD1-FLT3 CAR T cells in vitro and in vivo: Aim 1, further in vitro testing of scFvPD1-FLT3 CAR T cells; Aim 2, In vivo preclinical testing of scFvPD1- FLT3 CAR T cell efficacy; Aim 3, Combination therapy using scFvPD1-FLT3 CAR T cells and midostaurin, a drug recently approved by the U.S. FDA, which we have found induces FLT3 surface expression on AML blasts.