Innovative Cell Therapy for Pediatric Acute Myeloid Leukemia

摘要 儿童急性髓性白血病（pAML）的生存率落后于其他儿童白血病，这是由于 缺乏有效的pAML治疗。我们已经开发了一种新的T细胞疗法，LV-10，通过转导 正常外周血CD 4 + T淋巴细胞与表达IL 10的慢病毒载体。我们发现LV-10 可通过穿孔素和颗粒酶B溶解恶性髓系细胞。在测试的23个原代pAML原始细胞中，10个裂解 在所有测试的LV-10系中，6个是抗性的，7个具有可变的杀伤反应，这取决于LV-10系对 他们被暴露了。值得注意的是，死亡的6名患者的pAML原始细胞是耐药或耐药的。 抗LV-10裂解。pAML原始细胞对LV-10裂解的敏感性与其遗传学特征无关。 景观，但RNA测序（RNA-seq）的敏感和耐药的爆炸揭示了显着的差异 在他们的转录本中。pAML对裂解的抗性的分子机制尚不清楚; 阻力可以是：1.损害LV-10功能的活动过程，或2.由于缺乏抗原 对LV-10识别靶细胞所需的pAML。支持1.，RNA-seq显示表达增加 OX 40 L和CD 200在耐药pAML母细胞上的表达。它们各自的受体OX 40和CD 200 R1都是 在LV-10上表达，并且可以提供抑制性下游信号。因此，我们假设， pAML母细胞表达介导对LV-10裂解的抗性的蛋白质。然而，爆炸与变量 对杀伤的反应也表明，不同LV-10系的固有异质性可能会影响细胞的免疫应答。 裂解效率。因此，第二种假设指出，可变原始细胞的裂解需要LV-10和LV-20两者。 pAML具有允许表达的表型。 我们将通过确定裂解机制和pAML和LV-10特征来测试这些假设， LV-10杀伤，为优化LV-10在pAML中的临床应用铺平了道路。为此，我们将测试一种新的 45个pAML原始细胞的队列，用于它们对杀伤的敏感性以及它们对LV-10裂解能力和活化的影响。我们 我还将询问阻断OX 40 L和/或CD 200是否逆转耐药性，以及OX 40和CD 200 R1信号传导是否 改变LV-10的表型和功能（目的1）。在目标2中，我们将对敏感的， 在基线和相互作用后，对耐药和可变pAML和LV-10进行分析，以确定 pAML和LV-10群体异质性和基因表达，并揭示参与抗性的基因。 更广泛地说，我们注意到测试的pAML原始细胞的杀伤敏感性与传统风险无关 分层方法如WHO或FAB分类、细胞遗传学、年龄、性别、白细胞计数或 诱导后微小残留病（MRD）+/-。这表明，对细胞毒性杀伤的敏感性可能提供了一个 pAML患者分层的新方法。重要的是，IL-10的组成性分泌意味着同种异体LV-10 可在体内耐受，并且可作为容易获得的第三方现成产品而具有广泛应用 作为造血干细胞移植的一部分或作为pAML的独立细胞疗法施用。

ABSTRACT Survival for pediatric acute myeloid leukemia (pAML) has lagged behind other childhood leukemias due to the lack of effective pAML treatments. We have developed a novel T cell therapy, LV-10, made by transducing normal peripheral blood CD4+ T lymphocytes with a lentiviral vector expressing IL10. We discovered that LV-10 can lyse malignant myeloid cells via perforin and granzyme B. Of 23 primary pAML blast tested, 10 were lysed by all LV-10 lines tested, 6 were resistant , and 7 had a variable killing response depending on the LV-10 line to which they were exposed. Notably, the 6 patients who died had pAML blasts that were resistant or variably resistant to LV-10 lysis. The sensitivity of the pAML blasts to LV-10 lysis did not correlate with their genetic landscape, but RNA-sequencing (RNA-seq) of killing-sensitive and resistant blasts revealed dramatic differences in their transcriptomes. The molecular mechanism underlying the pAML resistance to lysis is yet unknown; resistance could be: 1. an active process impairing the LV-10 function, or 2. due to the absence of an antigen on pAML required for LV-10 recognition of target cells. In support of 1., RNA-seq revealed increased expression of OX40L and CD200 on resistant pAML blasts. Their respective receptors OX40 and CD200R1 are both expressed on LV-10, and may provide an inhibitory downstream signal. Thus, we hypothesize that resistant pAML blasts express protein(s) that mediate resistance to LV-10 lysis. However, the blasts with variable response to killing also suggest that the inherent heterogeneity of the different LV-10 lines may impact the efficiency of lysis. Thus, a second hypothesis states that lysis of variable blasts requires both LV-10 and pAML to have killing-permissive phenotypes. We will test these hypotheses by determining the lysis mechanism and pAML and LV-10 features required for LV-10 killing, paving the way for optimization of LV-10 for clinical use in pAML. To this end, we will test a new cohort of 45 pAML blasts for their sensitivity to killing, and their effect on LV-10 lytic capacity and activation. We will also ask if blocking OX40L and/or CD200 reverses the resistance, and if OX40 and CD200R1 signaling modifies LV-10 phenotype and function (Aim 1). In Aim 2, we will perform single cell RNA-seq on sensitive, resistant and variable pAML and LV-10, at baseline and after their interaction, to identify dynamic changes in both pAML and LV-10 population heterogeneity and gene expression, and uncover genes involved in resistance. More broadly, we note that the killing sensitivity of the tested pAML blasts did not correlate with conventional risk stratification methods such as WHO or FAB classification, cytogenetics, age, gender, leukocyte counts or minimal residual disease (MRD) +/- post-induction. This suggests that sensitivity to cytotoxic killing may offer a new means to stratify pAML patients. Importantly, the constitutive secretion of IL-10 implies allogeneic LV-10 can be tolerated in vivo, and may have wide application as a readily available, 3rd party off-the-shelf product administered as part of a hematopoietic stem cell transplantation, or as a stand-alone cell therapy forpAML.